Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms

ABSTRACT

The present invention provides novel eukaryotic DNA sequences coding for native protoporphyrinogen oxidase (protox) or modified forms of the enzyme which are herbicide tolerant. Plants having altered protox activity which confers tolerance to herbicides are also provided. These plants may be bred or engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or through increased levels of expression of the native protox gene, or they may be transformed with modified eukaryotic or prokaryotic protox coding sequences or wild type prokaryotic protox sequences which are herbicide tolerant. Diagnostic and other uses for the novel eukaryotic protox sequence are also described. Plant genes encoding wild-type and altered protox, purified plant protox, methods of isolating protox from plants, and methods of using protox-encoding genes are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/261,198 filed Jun. 16, 1994 now abandoned.

FIELD OF THE INVENTION

The invention relates generally to the manipulation of the enzymaticactivity responsible for the conversion of protoporphyrinogen IX toprotoporphyrin IX in a biosynthetic pathway common to all eukaryoticorganisms. In one aspect, the invention is applied to the development ofherbicide resistance in plants, plant tissues and seeds. In anotheraspect, the invention is applied to the development of diagnostics andtreatments for deficiencies in this enzymatic activity in animals,particularly humans.

BACKGROUND OF THE INVENTION

I. The Protox Enzyme and its Involvement in the Chlorophyll/HemeBiosynthetic Pathway

The biosynthetic pathways which leads to the production of chlorophylland heme share a number of common steps. Chlorophyll is a lightharvesting pigment present in all green photosynthetic organisms. Hemeis a cofactor of hemoglobin, cytochromes, P450 mixed-functionoxygenases, peroxidases, and catalases (see, e.g. Lehninger,Biochemistry. Worth Publishers, New York (1975)), and is therefore anecessary component for all aerobic organisms.

The last common step in chlorophyll and heme biosynthesis is theoxidation of protoporphyrinogen IX to protoporphyrin IX.Protoporphyrinogen oxidase (referred to herein as “protox”) is theenzyme which catalyzes this last oxidation step (Matringe et al.,Biochem. J. 260: 231 (1989)).

The protox enzyme has been purified either partially or completely froma number of organisms including the yeast Saccharomyces cerevisiae(Labbe-Bois and Labbe, In Biosynthesis of Heme and Chlorophyll, E. H.Dailey, ed. McGraw Hill: New York, pp. 235-285 (1990)), barleyetioplasts (Jacobs and Jacobs, Biochem. J. 244: 219 (1987)), and mouseliver (Dailey and Karr, Biochem. 26: 2697 (1987)). Genes encoding protoxhave been isolated from two prokaryotic organisms, Escherichia coli(Sasarrnan et al., Can. J. Microbiol. 39: 1155 (1993)) and Bacillussubtilis (Dailey et aL, J. Biol. Chem. 269: 813 (1994)). These genesshare no sequence similarity; neither do their predicted proteinproducts share any amino acid sequence identity. The E. coli protein isapproximately 21 kDa, and associates with the cell membrane. The B.subtilis protein is 51 kDa, and is a soluble, cytoplasmic activity.

Presently, too little is known about the protox enzyme to allowisolation of protox encoding genes from higher eukaryotic organisms(i.e. animals, plants and all other multicellular nucleate organismsother than lower eukaryotic microorganisms such as yeast, unicellularalgae, protozoans, etc.) using known approaches.

In particular, many of the standard techniques for isolation of newproteins and genes are based upon the assumption that they will besignificantly similar in primary structure (i.e. amino acid and DNAsequence) to known proteins and genes that have the same function. Suchstandard techniques include nucleic acid hybridization and amplificationby polymerase chain reaction using oligonucleotide primers correspondingto conserved amino acid sequence motifs. These techniques would not beexpected to be useful for isolation of eukaryotic protox genes usingpresent structural information which is limited to prokaryotic protoxgenes since there is no significant structural similarity even among theknown prokaryotic protox genes and proteins.

Another approach that has been used to isolate biosynthetic genes inother metabolic pathways from higher eukaryotes is the complementationof microbial mutants deficient in the activity of interest. For thisapproach, a library of cDNAs from the higher eukaryote is cloned in avector that can direct expression of the cDNA in the microbial host. Thevector is then transformed or otherwise introduced into the mutantmicrobe, and colonies are selected that are phenotypically no longermutant.

This strategy has worked for isolating genes from higher eukaryotes thatare involved in several metabolic pathways, including histidinebiosynthesis (e.g. U.S. patent application Ser. No. 08/061,644 to Wardet al., incorporated by reference herein in its entirety), lysinebiosynthesis (e.g. Frisch et al., Mol. Gen. Genet. 228: 287 (1991)),purine biosynthesis (e.g. Aimi et al., J. Biol. Chem. 265: 9011 (1990)),and tryptophan biosynthesis (e.g. Niyogi et al., Plant Cell 5: 1011(1993)). However, despite the availability of microbial mutants thoughtto be defective in protox activity (e.g. E. coli (Sasannan et al., J.Gen. Microbiol. 113: 297 (1979)), Salmonella typhimurium (Xu et al., J.Bacteriol. 174: 3953 (1992)), and Saccharomyces cerevisiae (Camadro etal,. Biochem. Biophys. Res. Comm. 106: 724 (1982)), application of thistechnique to isolate cDNAs encoding eukaryotic protox enzymatic activityis at best unpredictable based on the available information.

There are several reasons for this. First, the eukaryotic protox cDNAsequence may not be expressed at adequate levels in the mutant microbe,for instance because of codon usage inconsistent with the usagepreferences of the microbial host. Second, the primary translationproduct from the cloned eukaryotic coding sequence may not produce afunctional polypeptide, for instance if activity requires apost-translational modification, such as glycosylation, that is notcarried out by the microbe. Third, the eukaryotic protein may fail toassume its active conformation in the microbial host, for instance ifthe protein is normally targeted to a specific organellar membranesystem that the microbial host specifically lacks. This last possibilityis especially likely for the plant protox enzyme, which is associated inthe plant cell with organelles not present in microbial hosts used inthe complementation assay. In particular, the plant protox enzyme isassociated with both the chloroplast envelope and thylakoid membranes(Matringe et al., J. Biol. Chem. 267:4646 (1992)), and presumablyreaches those membrane systems as a result of a post-translationaltargeting mechanism involving both an N-terminal transit sequence, andintrinsic properties of the mature polypeptide (see, e.g. Kohorn andTobin, Plant Cell 1: 159 (1989); Li et al., Plant Cell 3: 709 (1991); Liet al., J. Biol. Chem. 267: 18999 (1992)).

II. Involvement of the Protox Gene in Animal/Human Disease Conditions

The protox enzyme is known to play a role in certain human diseaseconditions. Patients suffering from variegate porphyria, an autosomaldominant disorder characterized by both neuropsychiatric symptoms andskin lesions, have decreased levels of protox activity (Brenner andBloomer, New Engl. J. Med. 302: 765 (1980)). Due to the lack ofknowledge regarding the human protox enzyme and its corresponding gene,options for diagnosing and treating this disorder are presently verylimited.

III. The Protox Gene as a Herbicide Target

The use of herbicides to control undesirable vegetation such as weeds orplants in crops has become almost a universal practice. The relevantmarket exceeds a billion dollars annually. Despite this extensive use,weed control remains a significant and costly problem for farmers.

Effective use of herbicides requires sound management. For instance,time and method of application and stage of weed plant development arecritical to getting good weed control with herbicides. Since variousweed species are resistant to herbicides, the production of effectiveherbicides becomes increasingly important.

Unfortunately, herbicides that exhibit greater potency, broader weedspectrum and more rapid degradation in soil can also have greater cropphytotoxicity. One solution applied to this problem has been to developcrops which are resistant or tolerant to herbicides. Crop hybrids orvarieties resistant to the herbicides allow for the use of theherbicides without attendant risk of damage to the crop. Development ofresistance can allow application of a herbicide to a crop where its usewas previously precluded or limited (e.g. to pre-emergence use) due tosensitivity of the crop to the herbicide. For example, U.S. Pat. No.4,761,373 to Anderson et al. is directed to plants resistant to variousirnidazolinone or sulfonamide herbicides. The resistance is conferred byan altered acetohydroxyacid synthase (AHAS) enzyme. U.S. Pat. No.4,975,374 to Goodman et al. relates to plant cells and plants containinga gene encoding a mutant glutamine synthetase (GS) resistant toinhibition by herbicides that were known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,013,659 toBedbrook et al. is directed to plants that express a mutant acetolactatesynthase which renders the plants resistant to inhibition bysulfonylurea herbicides. U.S. Pat. No. 5,162,602 to Somers et al.discloses plants tolerant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The tolerance is conferred byan altered acetyl coenzyme A carboxylase(ACCase).

The protox enzyme serves as the target for a variety of herbicidalcompounds. The herbicides that inhibit protox include many differentstructural classes of molecules (Duke et al., Weed Sci. 39: 465 (1991);Nandihalli et al., Pesticide Biochem. Physiol. 43: 193 (1992); Matringeet al., FEBS Lett. 245: 35 (1989); Yanase and Andoh, Pesticide Biochem.Physiol. 35: 70 (1989)). These herbicidal compounds include thediphenylethers {e.g. acifluorfen,5-[2-chloro4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid; its methylester; or oxyfluorfen,2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles,(e.g. oxidiazon,3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic irnides (e.g. S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalirnide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and itsO-phenylpyrrolidino- and piperidinocarbamate analogs. Many of thesecompounds competitively inhibit the normal reaction catalyzed by theenzyme, apparently acting as substrate analogs.

The predicted mode of action of protox-inhibiting herbicides involvesthe accumulation of protoporphyrinogen IX in the chloroplast. Thisaccumulation is thought to lead to leakage of protoporphyrinogen IX intothe cytosol where it is oxidized by a peroxidase activity toprotoporphyrin IX. When exposed to light, protoporphyrin IX can causeformation of singlet oxygen in the cytosol. This singlet oxygen can inturn lead to the formation of other reactive oxygen species, which cancause lipid peroxidation and membrane disruption leading to rapid celldeath (Lee et al., Plant Physiol. 102: 881 (1993)).

Not all protox enzymes are sensitive to herbicides which inhibit plantprotox enzymes. Both of the protox enzymes encoded by genes isolatedfrom Escherichia coli (Sasarman et al., Can. J. Microbiol. 39: 1155(1993)) and Bacillus subtilis (Dailey et al., J. Biol. Chem. 269: 813(1994)) are resistant to these herbicidal inhibitors. In addition,mutants of the unicellular alga Chiamydomonas reinhardtii resistant tothe phenylimide herbicide S-23142 have been reported (Kataoka et al., J.Pesticide Sci. 15: 449 (1990); Shibata et al., In Research inPhotosynthesis, Vol.III, N. Murata, ed. Kluwer:Notherlands. pp. 567-570(1992)). At least one of these mutants appears to have an altered protoxactivity that is resistant not only to the herbicidal inhibitor on whichthe mutant was selected, but also to other classes of protox inhibitors(Oshio et al., Z. Naturforsch. 48c: 339 (1993); Sato et al., In ACSSymposium on Porphyric Pesticides, S. Duke, ed. ACS Press: Washington,D.C. (1994)). A mutant tobacco cell line has also been reported that isresistant to the inhibitor S-21432 (Che et al.,. Z. Naturforsch. 48c:350 (1993).

SUMMARY OF THE INVENTION

The present invention provides an isolated DNA molecule encoding theprotoporphyrinogen oxidase (protox) enzyme from a eukaryotic organism.In particular, the present invention provides isolated DNA moleculesencoding the protoporphyrinogen oxidase (protox) enzyme from a plant orhuman source.

Using the information provided by the present invention, the DNA codingsequence for the protoporphyrinogen oxidase (protox) enzyme from anyeukaryotic organism may be obtained using standard methods.

In accordance with these discoveries, the present invention providesplants, plant tissues and plant seeds with altered protox activity whichare resistant to inhibition by a herbicide at levels which normally areinhibitory to the naturally occurring protox activity in the plant.Plants encompassed by the invention include those which would bepotential targets for protox inhibiting herbicides, particularlyagronomically important crops such as maize and other cereal crops suchas wheat, oats, rye, sorghum, rice, barley, millet, turf and foragegrasses, and the like, as well as cotton, sugar cane, sugar beet,oilseed rape, and soybeans.

The present invention is directed further to methods for the productionof plants, plant tissues, and plant seeds which contain a protox enzymeresistant to, or tolerant of inhibition by a herbicide at aconcentration which inhibits the naturally occurring protox activity.One embodiment of the invention is directed to the preparation oftransgenic maize plants, maize tissue or maize seed which have beenstably transformed with a recombinant DNA molecule comprising a suitablepromoter functional in plants operably linked to a structural geneencoding an unmodified prokaryotic protox enzyme which is resistant tothe herbicide.

The invention is further directed to the preparation of transgenicplants, plant tissue and plant seed which has been stably transformedwith a recombinant DNA molecule comprising a suitable promoterfunctional in plants operably linked to a structural gene encoding anunmodified eukaryotic protox enzyme. This results in over-expression ofthe unmodified protox in the plant sufficient to overcome inhibition ofthe enzyme by the herbicide.

The present invention also embodies the production of plants whichexpress an altered protox enzyme tolerant of inhibition by a herbicideat a concentration which normally inhibits the activity of wild-type,unaltered protox. In this embodiment, the plant may be stablytransformed with a recombinant DNA molecule comprising a structural geneencoding the resistant protox, or prepared by direct selectiontechniques whereby herbicide resistant lines are isolated, characterizedand developed.

The present invention also embodies the recombinant production of theprotox enzyme; and methods for using recombinantly produced protox. Inparticular, the present invention provides methods of using purifiedprotox to screen for novel herbicides which affect the activity ofprotox, and to identify herbicide-resistant protox mutants. Genesencoding altered protox can be used as selectable markers in plant celltransformation methods.

The present invention is further directed to probes and methods fordetecting the presence and form of the protox gene and quantitatinglevels of protox transcripts in an organism. These methods may be usedto diagnose disease conditions which are associated with an altered formof the protox enzyme or altered levels of expression of the protoxenzyme.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention is directed to an isolated DNAmolecule which encodes a eukaryotic form of protoporphyrinogen oxidase(referred to herein as “protox”), the enzyme which catalyzes theoxidation of protoporphyrinogen IX to protoporphyrin IX. The DNA codingsequences and corresponding amino acid sequences for protox enzymes fromArabidopsis thaliana are provided as SEQ ID Nos. 1-4 and 9-10. The DNAcoding sequences and corresponding amino acid sequences for maize protoxenzymes are provided as SEQ ID Nos 5-8.

Any desired eukaryotic DNA encoding the protox enzyme may be isolatedaccording to the invention. One method taught for isolating a eukaryoticprotox coding sequence is represented by Example 1. In this method cDNAclones encoding a protox enzyme are identified from a library of cDNAclones derived from the eukaryote of interest based on their ability tosupply protox enzymatic activity to a mutant host organism deficient inthis activity. Suitable host organisms for use in this method are thosewhich can be used to screen cDNA expression libraries and for whichmutants deficient in protox activity are either available or can beroutinely generated. Such host organisms include, but are not limitedto, E. coli (Sasarrnan et al., J. Gen. Microbiol. 113: 297 (1979)),Salmonella typhimurium (Xu et al., J. Bacteriol. 174: 3953 (1992)), andSaccharomyces cerevisiae (Camadro et al. Biochem. Biophys. Res. Comm.106: 724 (1982)).

Alternatively, eukaryotic protox coding sequences may be isolatedaccording to well known techniques based on their sequence homology tothe Arabidopsis thaliana (SEQ ID Nos. 1,3 and 9) and Zea mays (SEQ IDNos. 5 and 7) protox coding sequences taught by the present invention.In these techniques all or part of the known protox coding sequence isused as a probe which selectively hybridizes to other protox codingsequences present in population of cloned genomic DNA fragments or cDNAfragments (i.e. genomic or cDNA libraries) from a chosen organism. Suchtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, e.g. Sambrook et al., MolecularCloning, eds., Cold Spring Harbor Laboratory Press. (1989)) andamplification by PCR using oligonucleotide primers corresponding tosequence domains conserved among known protox amino acid sequences (see,e.g. Innis et al., PCR Protocols, a Guide to Methods and Applicationseds., Academic Press (1990)). These methods are particularly well suitedto the isolation of protox coding sequences from organisms related tothe organism from which the probe sequence is derived. For example,application of these methods using the Arabidopsis or Zea mays codingsequence as a probe would be expected to be particularly well suited forthe isolation of protox coding sequences from other plant species.

The isolated eukaryotic protox sequences taught by the present inventionmay be manipulated according to standard genetic engineering techniquesto suit any desired purpose. For example, the entire protox sequence orportions thereof may be used as probes capable of specificallyhybridizing to protox coding sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique among protox coding sequences and arepreferably at least 10 nucleotides in length, and most preferably atleast 20 nucleotides in length. Such probes may be used to amplify andanalyze protox coding sequences from a chosen organism via the wellknown process of polymerase chain reaction (PCR). This technique may beuseful to isolate additional protox coding sequences from a desiredorganism or as a diagnostic assay to determine the presence of protoxcoding sequences in an organism and to associate altered codingsequences with particular adverse conditions such as autosomal dominantdisorder in humans characterized by both neuropsychiatric symptoms andskin lesions, have decreased levels of protox activity (Brenner andBloomer, New Engl. J. Med. 302: 765 (1980)).

Protox specific hybridization probes may also be used to map thelocation of the native eukaryotic protox gene(s) in the genome of achosen organism using standard techniques based on the selectivehybridization of the probe to genomic protox sequences. These techniquesinclude, but are not limited to, identification of DNA polymorphismsidentified or contained within the protox probe sequence, and use ofsuch polymorphisms to follow segregation of the protox gene relative toother markers of known map position in a mapping population derived fromself fertilization of a hybrid of two polymorphic parental lines (seee.g. Helentjaris et al., Plant Mol. Biol. 5: 109 (1985). Sommer et al.Biotechniques 12:82 (1992); D'Ovidio et al., Plant Mol. Biol. 15: 169(1990)). While any eukaryotic protox sequence is contemplated to beuseful as a probe for mapping protox genes from any eukaryotic organism,preferred probes are those protox sequences from organisms more closelyrelated to the chosen organism, and most preferred probes are thoseprotox sequences from the chosen organism. Mapping of protox genes inthis manner is contemplated to be particularly useful in plants forbreeding purposes. For instance, by knowing the genetic map position ofa mutant protox gene that confers herbicide resistance, flanking DNAmarkers can be identified from a reference genetic map (see, e.g.,Helentjaris, Trends Genet. 3: 217 (1987)). During introgression of theherbicide resistance trait into a new breeding line, these markers canthen be used to monitor the extent of protox-linked flanking chromosomalDNA still present in the recurrent parent after each round ofback-crossing.

Protox specific hybridization probes may also be used to quantitatelevels of protox mRNA in an organism using standard techniques such asNorthern blot analysis. This technique may be useful as a diagnosticassay to detect altered levels of protox expression that may beassociated with particular adverse conditions such as autosomal dominantdisorder in humans characterized by both neuropsychiatric symptoms andskin lesions, have decreased levels of protox activity (Brenner andBloomer, New Engl. J. Med. 302: 765 (1980)).

For recombinant production of the enzyme in a host organism, the protoxcoding sequence may be inserted into an expression cassette designed forthe chosen host and introduced into the host where it is recombinantlyproduced. The choice of specific regulatory sequences such as promoter,signal sequence, 5′ and 3′ untranslated sequences, and enhancer, iswithin the level of skill of the routineer in the art. The resultantmolecule, containing the individual elements linked in proper readingframe, may be inserted into a vector capable of being transformed intothe host cell. Suitable expression vectors and methods for recombinantproduction of proteins are well known for host organisms such as E. coli(see, e.g. Studier and Moffatt, J. Mol. Biol. 189: 113 (1986); Brosius,DNA 8: 759 (1989)), yeast (see, e.g., Schneider and Guarente, Meth.Enzymol. 194: 373 (1991)) and insect cells (see, e.g., Luckow andSummers, Bio/Technol. 6: 47 (1988)). Specific examples include plasmidssuch as pBluescript (Stratagene, La Jolla, Calif.), PFLAG (InternationalBiotechnologies, Inc., New Haven, Conn.), pTrcHis (Invitrogen, La Jolla,Calif.), and baculovirus expression vectors, e.g., those derived fromthe genome of Autographica californica nuclear polyhedrosis virus(AcMNPV). A preferred baculovirus/insect system is pVl 11392/Sf21 cells(Invitrogen, La Jolla, Calif.).

Recombinantly produced eukaryotic protox enzyme is useful for a varietyof purposes. For example, it may be used to supply protox enzymaticactivity in vitro. It may also be used in an in vitro assay to screenknown herbicidal chemicals whose target has not been identified todetermine if they inhibit protox. Such an in vitro assay may also beused as a more general screen to identify chemicals which inhibit protoxactivity and which are therefore herbicide candidates. Alternatively,recombinantly produced protox enzyme may be used to further characterizeits association with known inhibitors in order to rationally design newinhibitory herbicides as well as herbicide tolerant forms of the enzyme.

Typically, the inhibitory effect on protox is determined by measuringfluorescence at about 622 to 635 nm, after excitation at about 395 to410 nM (see, e.g. Jacobs and Jacobs, Enyzme 28: 206 (1982); Sherman etal., Plant Physiol. 97: 280 (1991)). This assay is based on the factthat protoporphyrin IX is a fluorescent pigment, and protoporphyrinogenIX is nonfluorescent. Protein extracts are prepared from selectedsubcellular fractions, e.g. etioplasts, mitochondria, microsomes, orplasma membrane, by differential centrifugation (see, e.g. Lee et al.,Plant Physiol. 102:881 (1993); Prado et al, Plant Physio. 65: 956(1979); Jackson and Moore, in Plant Organelles. Reid, ed., pp. 1-12;Jacobs and Jacobs, Plant Physiol. 101: 1181 (1993)). Protoporphyrinogenis prepared by reduction of protoporphyrin with a sodium amalgam asdescribed by Jacobs and Jacobs (1982). Reactions mixtures typicallyconsist of 100 mM Hepes (pH 7.5), 5 mM EDTA, 2 mM DTT, about 2 μMprotoporphyrinogen IX, and about 1 mg/mL protein extract. Inhibitorsolutions in various concentrations, e.g. 1 mM, 100 uM, 10 uM, 1 uM, 100nM, 10 nM, 1 nM, 100 pM, are added to the enzyme extract prior to theinitiation of the enzyme reaction. Once the protein extract is added,fluorescence is monitored for several minutes, and the slope of theslope (reaction rate) is calculated from a region of linearity. IC₅₀ isdetermined by comparing the slope of the inhibited reaction to a controlreaction.

Another embodiment of the present invention involves the use of protoxin an assay to identify inhibitor-resistant protox mutants. A typicalassay is as follows:

(a) incubating a first sample of protox and its substrate,protoporphyrinogen IX, in the presence of a second sample comprising aprotox inhibitor;

(b) measuring the enzymatic activity of the protox from step (a);

(c) incubating a first sample of a mutated protox and its substrate inthe presence of a second sample comprising the same protox inhibitor;

(d) measuring the enzymatic activity of the mutated protox from step(c); and

(e) comparing the enzymatic activity of the mutated protox with thatprovided by the unmutated protox.

The reaction mixture and the reaction conditions are the same as for theassay to identify inhibitors of protox (inhibitor assay) with thefollowing modifications. First, a protox mutant, obtained as describedabove, is substituted in one of the reaction mixtures for the wild-typeprotox of the inhibitor assay. Second, an inhibitor of wild-type protoxis present in both reaction mixtures. Third, mutated activity (enzymeactivity in the presence of inhibitor and mutated protox) and unmutatedactivity (enzyme activity in the presence of inhibitor and wild-typeprotox) are compared to determine whether a significant increase inenzyme activity is observed in the mutated activity when compared to theunmutated activity. Mutated activity is any measure of enzymaticactivity of the mutated protox enzyme while in the presence of asuitable substrate and the inhibitor. Unmutated activity is any measureof enzymatic activity of the wild-type protox enzyme while in thepresence of a suitable.substrate and the inhibitor. A significantincrease is defined as an increase in enzymatic activity that is largerthan the margin of error inherent in the measurement technique,preferably an increase by about 2-fold of the activity of the wild-typeenzyme in the presence of the inhibitor, more preferably an increase byabout 5-fold, most preferably an increase greater than by about 10-fold.

The herbicides that inhibit protox include many different structuralclasses of molecules (Duke et al., Weed Sci. 39: 465 (1991); Nandihalliet al., Pesticide Biochem. Physiol. 43: 193 (1992); Matringe et al.,FEBS Lett. 245: 35 (1989); Yanase and Andoh, Pesticide Biochem. Physiol.35: 70 (1989)), including the diphenylethers {e.g. acifluorifen,5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid; its methylester; or oxyfluorfen,2-chloro-1-(3-ethoxy4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles(e.g. oxidiazon,3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one),cyclic imides (e.g. S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalirnide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and itsO-phenylpyrrolidino- and piperidinocarbamate analogs.

The diphenylethers of particular significance are those having thegeneral formula

wherein R equals —COONa (Formula II), —CONHSO₂CH₃ (Formula III) or—COOCH₂COOC₂H₅ (Formula IV; see Maigrot et al., Brighton Crop ProtectionConference-Weeds: 47-51 (1989)). Additional diphenylethers of interestare those where R equals:

(Fornula IVa; see Hayashi et al., Brighton Crop ProtectionConference-Weeds: 53-58 (1989)). An additional diphenylether of interestis one having the formula:

(Formula IVb; bifenox, see Dest et al., Proc. Northeast Weed Sci. Conf.27: 31 (1973)).

Also of significance are the class of herbicides known as irnides,having the general formula

wherein Q equals

(see Hemper et al. (1995) in “Proceedings of the Eighth InternationalCongress of Pesticide Chemistry”, Ragdale et al., eds., Amer. Chem. Soc,Washington, D.C., pp.42-48 (1994));

and R₁ equals H, Cl or F, R₂ equals Cl and R₃ is an optimallysubstituted ether, thioether, ester, amino or alkyl group.Alternatively, R₂ and R₃ together may form a 5 or 6 memberedheterocyclic ring. Examples of imide herbicides of particular interestare

The herbicidal activity of the above compounds is described in theProceedings of the 1991 Brighton Crop Protection Conference, Weeds(British Crop Protection Council) (Formulae X and XVI), Proceedings ofthe 1993 Brighton Crop Protection Conference, Weeds (British CropProtection Council) (Formulae XII and XIII), U.S. Pat. No. 4,746,352(Formula XI) and Abstracts of the Weed Science Society of America vol.33, pg. 9 (1993)(Formula XIV).

The most preferred imide herbicides are those classified as aryluracilsand having the general formula

wherein R signifies the group (C₂₋₆-alkenyloxy)carbonyl-C₁₋₄-alkyl, asdisclosed in U.S. Pat. No. 5,183,492, herein incorporated by reference.

Also of significance are herbicides having the general formula:

N-substituted pyrazoles of the general formula:

wherein R₁ is C₁-C₄-alkyl, optionally substituted by one or more halogenatoms;

R₂ is hydrogen, or a C₁-C₄-alkoxy, each of which is optionallysubstituted by one or more halogen atoms, or

R₁ and R₂ together from the group —(CH₂)_(n)—X—, where X is bound at R₂;

R₃ is hydrogen or halogen,

R₄ is hydrogen or C₁-C₄-alkyl,

R₅ is hydrogen, nitro, cyano or the group —COOR₆ or —CONR₇R₈, and

R₆ is hydrogen, C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl; (seeinternational patent publications WO 94/08999, WO 93/10100, and U.S.Pat. No. 5,405,829 assigned to Schering);

N-phenylpyrazoles, such as:

and 3-substituted-2-aryl-4,5,6,7-tetrahydroindazoles (Lyga et al.Pesticide Sci. 42:29-36 (1994)).

Levels of herbicide which normally are inhibitory to the activity ofprotox include application rates known in the art, and which dependpartly on external factors such as environment, time and method ofapplication. For example, in the case of the imide herbicidesrepresented by Formulae V through IX, and more particularly thoserepresented by Formulae X through XVII, the application rates range from0.0001 to 10 kg/ha, preferably from 0.005 to 2 kg/ha. This dosage rateor concentration of herbicide may be different, depending on the desiredaction and particular compound used, and can be determined by methodsknown in the art.

The present invention is further directed to plants, plant tissue andplant seeds tolerant to herbicides that inhibit the naturally occurringprotox activity in these plants, wherein the tolerance is conferred byan altered protox enzyme activity. Representative plants include anyplants to which these herbicides are applied for their normally intendedpurpose. Preferred are agronomically important crops, i.e., angiospermsand gymnosperms significant as cotton, soya, rape sugar beet, maize,rice, wheat, barley, oats, rye, sorghum, millet, turf, forage, turfgrasses and the like.

By “altered protox enzyme activity” is meant a protox enzymatic activitydifferent from that which naturally occurs in a plant (i.e. protoxactivity which occurs naturally in the absence of direct or indirectmanipulation of such activity by man) which is resistant to herbicidesthat inhibit the naturally occurring activity. Altered protox enzymeactivity may be conferred upon a plant according to the invention byincreasing expression of wild-type, herbicide-sensitive protox,expressing an altered, herbicide-tolerant eukaryotic protox enzyme inthe plant, expressing an unmodified or modified bacterial form of theprotox enzyme which is herbicide resistant in the plant, or by acombination of these techniques.

Achieving altered protox enzyme activity through increased expressionresults in a level of protox in the plant cell at least sufficient toovercome growth inhibition caused by the herbicide. The level ofexpressed protox generally is at least two times, preferably five times,and more preferably at least ten times the natively expressed amount.Increased expression may be due to multiple copies of a wild-type protoxgene; multiple occurrences of the protox coding sequence within theprotox gene (i.e. gene amplification) or a mutation in the non-coding,regulatory sequence of the endogenous protox gene in the plant cell.Plants containing such altered protox enzyme activity can be obtained bydirect selection in plants. This method is known in the art. See, e.g.Somers et al. in U.S. Pat. No. 5,162,602, and Anderson et al. in U.S.Pat. No. 4,761,373, and references cited therein. These plants also maybe obtained via genetic engineering techniques known in the art.Increased expression of herbicide-sensitive protox also can beaccomplished by stably transforming a plant cell with a recombinant orchimeric DNA molecule comprising a promoter capable of drivingexpression of an associated structural gene in a plant cell, linked to ahomologous or heterologous structural gene encoding protox. By“homologous,” it is meant that the protox gene is isolated from anorganism taxonomically identical to the target plant cell. By“heterologous,” it is meant that the protox gene is obtained from anorganism taxonomically distinct from the target plant cell. Homologousprotox genes can be obtained by complementing a bacterial or yeastauxotrophic mutant with a cDNA expression library from the target plant.See, e.g. Example 1 and Snustad et al, Genetics 120:1111-1114 (1988)(maize glutamine synthase); Delauney et al., Mol. Genet. 221:299-305(1990) (soybean -pyrroline -5-carboxylate reductase); Frisch et al.,Mol. Gen. Genet. 228:287-293(1991) (maize dihydrodipicolinate synthase);Eller et al., Plant Mol. Biol. 18:557-566 (1992) (rape chloroplast3-isopropylmalate dehydrogenase); Proc. Natl. Acad. Sci, USA88:1731-1735 (1991); Minet et al., Plant J. 2:417-422 (1992)(dihydroorotate dehydrogenase) and references cited therein. Other knownmethods include screening genomic or cDNA libraries of higher plants,for example, for sequences that cross-hybridize with specific nucleicacid probes, or by screening expression libraries for the production ofprotox enzymes that cross-react with specific antibody probes. Apreferred method involves complementing an E. coli hemG auxotrophicmutant with a maize or Arabidopsis thaliana cDNA library.

Examples of promoters capable of functioning in plants or plant cells,i.e., those capable of driving expression of the associated structuralgenes such as protox in plant cells, include the cauliflower mosaicvirus (CaMV) 19S or 35S promoters and CAMV double promoters; nopalinesynthase promoters; pathogenesis-related (PR) protein promoters; smallsubunit of ribulose bisphosphate carboxylase (ssuRUBISCO) promoters, andthe like. Preferred are the rice actin promoter (McElroy et al., Mol.Gen. Genet. 231: 150 (1991)), maize ubiquitin promoter (EP 0 342 926;Taylor et al., Plant Cell Rep.12: 491 (1993)), and the Pr-1 promoterfrom tobacco, Arabidopsis, or maize (see U.S. patent application Ser.No. 08/181,271 to Ryals et al., incorporated by reference herein in itsentirety). Also preferred are the 35S promoter and an enhanced or double35S promoter such as that described in Kay et al., Science 236:1299-1302 (1987) and the double 35S promoter cloned into pCGN2113,deposited as ATCC 40587, which are disclosed in each of commonly ownedcopending application Ser. No. 07/580,431, filed Sep. 7, 1990, which isa continuation-in-part of Ser. No. 07/425,504, filed Oct. 20, 1989,which is a continuation-in-part of Ser. No. 07/368,672, filed Jun. 20,1989, which is a continuation-in-part of Ser. No. 07/329,018, filed Mar.24, 1989, the relevant disclosures of which are herein incorporated byreference in their entirety. The promoters themselves may be modified tomanipulate promoter strength to increase protox expression, inaccordance with art-recognized procedures.

Signal or transit peptides may be fused to the protox coding sequence inthe chimeric DNA constructs of the invention to direct transport of theexpressed protox enzyme to the desired site of action. Examples ofsignal peptides include those natively linked to the plantpathogenesis-related proteins, e.g. PR-1, PR-2, and the like. See, e.g.,Payne et al., Plant Mol. Biol. 11:89-94 (1988). Examples of transitpeptides include the chloroplast transit peptides such as thosedescribed in Von Heijne et al., Plant Mol. Biol. Rep. 9: 104-126 (1991);Mazur et al., Plant Physiol. 85: 1110 (1987); Vorst et al., Gene 65: 59(1988), and mitochondrial transit peptides such as those described inBoutry et al., Nature 328:340-342 (1987). Chloroplast and mitochondrialtransit peptides are contemplated to be particularly useful with thepresent ivention as protox enzymatic activity typically occurs withinthe mitochondria and chloroplast. Most preferred for use are chloroplasttransit peptides as inhibition of the protox enzymatic activity in thechloroplasts is contemplated to be the primary basis for the action ofprotox-inhibiting herbicides (Witkowski and Halling, Plant Physiol. 87:632 (1988); Lehnen et al., Pestic. Biochem. Physiol. 37: 239 (1990);Duke et al., Weed Sci. 39: 465 (1991)). Also included are sequences thatresult in localization of the encoded protein to various cellularcompartments such as the vacuole. See, for example, Neuhaus et al.,Proc. Natl. Acad. Sci. USA 88: 10362-10366 (1991) and Chrispeels, Ann.Rev. Plant Physiol. Plant Mol. Biol. 42: 21-53 (1991). The relevantdisclosures of these publications are incorporated herein by referencein their entirety.

The chimeric DNA construct(s) of the invention may contain multiplecopies of a promoter or multiple copies of the protox structural genes.In addition, the construct(s) may include coding sequences for markersand coding sequences for other peptides such as signal or transitpeptides, each in proper reading frame with the other functionalelements in the DNA molecule. The preparation of such constructs arewithin the ordinary level of skill in the art.

Useful markers include peptides providing herbicide, antibiotic or drugresistance, such as, for example, resistance to hygromycin, kanamycin,G418, gentamycin, lincomycin, methotrexate, glyphosate,phosphinothricin, or the like. These markers can be used to select cellstransformed with the chimeric DNA constructs of the invention fromuntransformed cells. Other useful markers are peptidic enzymes which canbe easily detected by a visible reaction, for example a color reaction,for example luciferase, β-glucuronidase, or β-galactosidase.

Altered protox enzyme activity may also be achieved through thegeneration or identification of modified forms of the isolatedeukaryotic protox coding sequence having at least one amino acidsubstitution, addition or deletion which encode an altered protox enzymeresistant to a herbicide that inhibits the unaltered, naturally occuringform (i.e. forms which occur naturally in a eukaryotic organism withoutbeing manipulated, either directly via recombinant DNA methodology orindirectly via selective breeding, etc., by man). Genes encoding suchenzymes can be obtained by numerous strategies known in the art. A firstgeneral strategy involves direct or indirect mutagenesis procedures onmicrobes. For instance, a genetically manipulable microbe, e.g. E. colior S. cerevisiae, may be subjected to random mutagenesis in vivo, with,for example UV light or ethyl or methyl methane sulfonate. Mutagenesisprocedures are described, for example in Miller, Experiments inMolecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1972); Davis et al., Advanced Bacterial Genetics, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1980); Sherman et al.,Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1983); and U.S. Pat. No.4,975,374 (Goodman et al). Themicrobe selected for mutagenesis contains a normally herbicide sensitiveeukaryotic protox gene and is dependent upon the protox activityconferred by this gene. The mutagenized cells are grown in the presenceof the herbicide at concentrations which inhibit the unmodified protoxenzyme. Colonies of the mutagenized microbe that grow better than theunmutagenized microbe in the presence of the inhibitor (i.e. exhibitresistance to the inhibitor) are selected for further analysis. Theprotox genes from these colonies are isolated, either by cloning or bypolymerase chain reaction amplification, and their sequences elucidated.Sequences encoding an altered protox enzyme are then cloned back intothe microbe to confirm their ability to confer inhibitor resistance.

A second method of obtaining mutant herbicide-resistant alleles of theeukaryotic protox enzyme involves direct selection in plants. Forexample, the effect of a protox inhibitor such those as described above,on the growth inhibition of plants such as Arabidopsis, soybean, ormaize may be determined by plating seeds sterilized by art-recognizedmethods on plates on a simple minimal salts medium containing increasingconcentrations of the inhibitor. Such concentrations are in the range of0.001,0.003, 0.01, 0.03,0.1,0.3, 1,3, 10,30, 110,300, 1000 and 3000parts per million (ppm). The lowest dose at which significant growthinhibition can be reproducibly detected is used for subsequentexperiments.

Mutagenesis of plant material may be utilized to increase the frequencyat which resistant alleles occur in the selected population. Mutagenizedseed material can be derived from a variety of sources, includingchemical or physical mutagenesis or seeds, or chemical or physicalmutagenesis or pollen (Neuffer, In Maize for Biological Research.Sheridan, ed. Univ.Press, Grand Forks, N.Dak., pp. 61-64 (1982)), whichis then used to fertilize plants and the resulting M₁ mutant seedscollected. Typically, for Arabidopsis, M₂ seeds (Lehle Seeds, Tucson,Ariz.), i.e. progeny seeds of plants grown from seeds mutagenized withchemicals, such as ethyl methane sulfonate, or with physical agents,such as gamma rays or fast neutrons, are plated at densities of up to10,000 seeds/plate (10 cm diameter) on minimal salts medium containingan appropriate concentration of inhibitor to select for resistance.Seedlings that continue to grow and remain green 7-21 days after platingare transplanted to soil and grown to maturity and seed set. Progeny ofthese seeds are tested for resistance to the herbicide. If theresistance trait is dominant, plants whose seed segregate3:1::resistant:sensitive are presumed to have been heterozygous for theresistance at the M₂ generation. Plants that give rise to all resistantseed are presumed to have been homozygous for the resistance at the M₂generation. Such mutagenesis on intact seeds and screening of their M2progeny seed can also be carried out on other species, for instancesoybean (see,e.g. U.S. Pat. No. 5,084,082 (Sebastian)). Mutant seeds tobe screened for herbicide tolerance can also be obtained as-a result offertilization with pollen mutagenized by chemical or physical means.

Two approaches can be taken to confirm that the genetic basis of theresistance is an altered protox gene. First, alleles of the protox genefrom plants exhibiting resistance to the inhibitor can be isolated usingPCR with primers based either upon conserved regions in the Arabidopsisand maize protox cDNA sequences shown in SEQ ID NOS: 1,3,5,7 below or,more preferably, based upon the unaltered protox gene sequences from theplant used to generate resistant alleles. After sequencing the allelesto determine the presence of mutations in the coding sequence, thealleles can be tested for their ability to confer resistance to theinhibitor on plants into which the putative resistance-conferringalleles have been transformed. These plants can be either Arabidopsisplants or any other plant whose growth is susceptible to the inhibitors.Second, the protox genes can be mapped relative to known restrictionfragment length polymorphisms (RFLPs) (See, for example, Chang et al.Proc. Natl. Acad, Sci, USA 85:6856-6860 (1988); Nam et al., Plant Cell1:699-705 (1989). The resistance trait can be independently mapped usingthe same markers. If resistance is due to a mutation in that protoxgene, the resistance trait will map to a position indistinguishable fromthe position of a protox gene.

A third method of obtaining herbicide-resistant alleles of protox is byselection in plant cell cultures. Explants of plant tissue, e.g.embryos, leaf disks, etc. or actively growing callus or suspensioncultures of a plant of interest are grown on defined medium lacking hemein the presence of increasing concentrations of the inhibitory herbicideor an analogous inhibitor suitable for use in a laboratory environment.Varying degrees of growth are recorded in different cultures. In certaincultures, fast-growing variant colonies arise that continue to grow evenin the presence of normally inhibitory concentrations of inhibitor. Thefrequency with which such faster-growing variants occur can be increasedby treatment with a chemical or physical mutagen before exposing thetissues or cells to the herbicide. Putative resistance-conferringalleles of the protox gene are isolated and tested as described in theforegoing paragraphs. Those alleles identified as conferring herbicideresistance may then be engineered for optimal expression and transformedinto the plant. Alternatively, plants can be regenerated from the tissueor cell cultures containing these alleles.

A fourth method involves mutagenesis of wild-type, herbicide sensitiveprotox genes in bacteria or yeast, followed by culturing the microbe onmedium that lacks heme, but which contains inhibitory concentrations ofthe inhibitor and then selecting those colonies that grow in thepresence of the inhibitor. More specifically, a plant cDNA, such as theArabidopsis or maize cDNA encoding protox is cloned into a microbe thatotherwise lacks protox activity. Examples of such microbes include E.coli, S. typhimurium, and S. cerevisiae auxotrophic mutants, includingE. coli strain SASX38 (Sasarman et al., J. Gen. Microbiol. 113: 297(1979), S. typhimurium strain TE2483 or TT13680 (Xu et al., J.Bacteriol. 174: 3953 (1992)), and the hem14-1 yeast mutant (Camadro etal., Biochem. Biophys. Res. Comm. 106: 724 (1982)). The transformedmicrobe is then subjected to in vivo mutagenesis such as describedimmediately above, or to in vitro mutagenesis by any of several chemicalor enzymatic methods known in the art, e.g. sodium bisulfite (Shortle etal., Methods Enzymol. 100:457-468 (1983); methoxylamine (Kadonaga etal., Nucleic Acids Res. 13:1733-1745 (1985); oligonucleotide-directedsaturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA,83:710-714 (1986); or various polymerase misincorporation strategies(see, e.g. Shortle et al., Proc. Natl. Acad. Sci. USA, 79:1588-1592(1982); Shiraishi et al., Gene 64:313-319 (1988); and Leung et al.,Technique 1:11-15 (1989). Colonies that grow in the presence of normallyinhibitory concentrations of inhibitor are picked and purified byrepeated restreaking. Their plasmids are purified and tested for theability to confer resistance to the inhibitor by retransforming theminto the protox-lacking microbe. The DNA sequences of protox cDNAinserts from plasmids that pass this test are then determined.

Once a herbicide resistant protox allele is identified, it may begenetically engineered for optimal expression in a crop plant. This mayinclude altering the coding sequence of the resistance allele foroptimal expression in the crop species of interest. Methods formodifying coding sequences to achieve optimal expression in a particularcrop species are well known (see, e.g. Perlak et al., Proc. Natl. Acad.Sci. USA 88: 3324 (1991); Koziel et al., Bio/technol. 11: 194 (1993)).Genetically engineering the protox allele for optimal expression mayalso include operably linking the appropriate regulatory sequences (i.e.promoter, signal sequence, transcriptional terminators). Preferredpromoters will be those which confer high level constitutive expressionor, more preferably, those which confer specific high level expressionin the tissues susceptible to damage by the herbicide.

The recombinant DNA molecules can be introduced into the plant cell in anumber of art-recognized ways. Those skilled in the art will appreciatethat the choice of method might depend on the type of plant, i.e.monocot or dicot, targeted for transformation. Suitable methods oftransforming plant cells include microinjection (Crossway et al.,BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Proc.Natl. Acad. Sci. USA 83:5602-5606 (1986), Agrobacterium mediatedtransformation (Hinchee et al., Biotechnology 6:915-921 (1988)), directs gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), andballistic particle acceleration using devices available from Agracetus,Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al.,Biotechnology 6:923-926 (1988)). Also see, Weissinger et al., AnnualRev. Genet. 22:421477 (1988); Sanford et al., Particulate Science andTechnology 5:27-37 (1987)(onion); Christou et al., Plant Physiol.87:671-674 (1988)(soybean); McCabe et al., Bio/Technology 6:923-926(1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rice);Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize);Klein et al., Bio/Technology 6:559-563 (1988)(maize); Klein et al.,Plant Physiol. 91:440-444 (1988)(maize); Fromm et al., Bio/Technology8:833-839 (1990); and Gordon-Kanim et al., Plant Cell 2:603-618(1990)(maize).

Where a herbicide resistant protox allele is obtained via directselection in a crop plant or plant cell culture from which a crop plantcan be regenerated, it may be moved into commercial varieties usingtraditional breeding techniques to develop a herbicide tolerant cropwithout the need for genetically engineering the allele and transformingit into the plant. Alternatively, the herbicide resistant allele may beisolated, genetically engineered for optimal expression and thentransformed into the desired variety.

Genes encoding altered protox resistant to a protox inhibitor can alsobe used as selectable markers in plant cell transformation methods. Forexample, plants, plant tissue or plant cells transformed with atransgene can also be transformed with a gene encoding an altered protoxcapable of being expressed by the plant. The thus-transformed cells aretransferred to medium containing the protox inhibitor wherein only thetransformed cells will survive. Protox inhibitors contemplated to beparticularly useful as selective agents are the diphenylethers {e.g.acifluorfen, 5-[2-chloro4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid;its methyl ester; or oxyfluorfen,2-chloro-1-(3-ethoxy4-nitrophenoxy)4(trifluorobenzene)}, oxidiazoles,(e.g. oxidiazon,3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one, cyclic imides (e.g. S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopyiate and itsO-phenylpyrrolidino- and piperidinocarbamate analogs. The method isapplicable to any plant cell capable of being transformed with analtered protox-encoding gene, and can be used with any transgene ofinterest. Expression of the transgene and the protox gene can be drivenby the same promoter functional on plant cells, or by separatepromoters.

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by T. Maniatis, E. F. Fritschand J. Sambrook. Molecular Cloning: A Laboratory manual, Cold SpringHarbor laboratory, Cold Spring Harbor, N.Y. (1982) and by T. J. Silhavy,M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).

Example 1 Isolation of Arabidopsis cDNAs Encoding Protox Genes byFunctional Complementation of an E. coli Mutant.

An Arabidopsis thaliana (Landsberg) cDNA library in the plasmid vectorpFL61 (Minet et al., Plant J. 2:417-422 (1992)) was obtained andamplified. A second Arabidopsis (Columbia) cDNA library in the UniZaplambda vector (Stratagene) was purchased and amplified as pBluescriptplasmids by mass in vivo excision of the phage stock. The E. coli hemGmutant SAS×38 (Sasarman et al., J. Gen. Microbiol. 113: 297 (1979)) wasobtained and maintained on L media containing 20 mg/ml hematin (UnitedStates Biochemicals). The plasmid libraries were transformed into SAS×38by electroporation using the Bio-Rad Gene Pulser and the manufacturer'sconditions. The cells were plated on L agar containing 100 mg/mlampicillin at a density of approximately 500,000 transformants/10 cmplate. The cels were incubated at 37° C. for 40 hours in low light andselected for the ability to grow without the addition of exogenous heme.Heme prototrophs were recovered at a frequency of 400/10⁷ from the pFL61library and at a frequency of 2/10⁷ from the pBluescript library.Plasmid DNA was isolated from 24 colonies for sequence analysis. Each ofthe 24 was retransformed into SAS×38 to verify ability to complement.

Sequence analysis revealed two classes of putative protox clones. Ninewere of the type designated “Protox-1.” Each was derived from the samegene, and two were full-length clones. The cDNA is 1719bp in length andencodes a protein of molecular weight 57.7 kDa. The N-terminal peptidesequence has features characteristic of a chloroplast transit peptide ofapproximately 60 amino acids. A database search with the GAP program(Deveraux et al., Nucleic Acids Res. 12:387-395 (1984) reveals homologywith the B. subtilis hemY (protox) protein (Hansson and Hederstedt 1992,Dailey et al., J. Biol. Chem. 269: 813 (1994)). The two proteins are 53%similar, 31% identical with regions of high homology, including theproposed dinucleotide binding domain of the hemY protein (Dailey et al.,J. Biol. Chem. 269: 813 (1994)).

The other 15 cDNA clones were of the type designated “Protox-2”. Thesealso appeared to arise from a single gene. The apparently full-lengthcDNA is 1738bp in length and encodes a protein of molecular weight55.6kD. The amino terminus is somewhat characteristic of a mitochondrialtransit peptide. The Protox-2 protein has limited homology to Protox-1(53% similar, 28% identical) and to the B. subtilis protox (50% similar,27% identical).

Protox-1, in the pBluescript SK vector, was deposited Apr. 5, 1994 aspWDC-2 (NRRL #B-21238).

Protox-2, in the pFL61 vector, was deposited Apr. 5, 1994 as pWDC-1(NRRL #B-21237).

The Arabidopsis cDNA encoding protox-1 contained in pWDC-2 and protox-2contained in pWDC-1 are set forth in SEQ ID NOS:1 and 3, respectively,below.

Example 2 Isolation of Maize cDNAs Encoding Protox Genes by FunctionalComplementation of an E. coli Mutant.

A Zea Mays (B73 inbred) cDNA library in lambda UniZap was purchased fromStratagene and converted to a pBluescript library by mass in vivoexcision. A second custom-made UniZap maize cDNA library was purchasedfrom Clontech, and similarly converted to pBluescript plasmids.Selection for functional protox genes from maize was just as describedfor the Arabidopsis libraries above in Example 1.

Two heme prototrophs in 107 transformants were isolated from theStratagene library, shown to recomplement and sequenced. These cDNAswere identical and proved to be homologs of Arabidopsis Protox-1. Thismaize clone, designated MzProtox-1, is incomplete. The cDNA is 1698bp inlength and codes only for the putative mature protox enzyme; there is notransitpeptide sequence and no initiating methionine codon. The gene is68% identical to Arab Protox-1 at the nucleotide level and 78% identical(87% similar) at the amino acid level (shown in Table 1).

A single heme prototroph in 10⁷ transformants was obtained from theClontech library, shown to recomplement, and sequenced. The cDNA appearsto be complete, is 2061 bp in length and encodes a protein of 59 kDa.This clone is a maize homolog of Arabidopsis Protox-2 and is designatedMzProtox-2. The gene is 58% identical to Arab Protox-2 at the nucleotidelevel and 58% identical (76% similar) at the amino acid level (shown inTable 2). The maize clone has an N-terminal sequence that is 30 aminoacids longer than the Arabidopsis clone. As with the Arabidopsis clones,homology between the two maize protox genes is quite low, with only 31%identity between the two protein sequences.

MzProtox-1, in the pBluescript SK vector, deposited May 20, 1994 aspWDC-4 with the NRRL (#B-21260), shown in SEQ ID NO:5.

MzProtox-2, in the pBluescript SK vector, deposited May 20, 1994 aspWDC-3 with the NRRL (#B-21259), shown in SEQ ID NO:7.

Example 3 Isolation of Additional Protox Genes Based on SequenceHomology to Known Protox Coding Sequences

A phage or plasmid library is plated at a density of approximately10,000 plaques on a 10 cm Petri dish, and filter lifts of the plaquesare made after overnight growth of the plants at 37 C. The plaque liftsare probed with one of the cDNAs set forth in SEQ ID NOS:1, 3, 5 or 7,labeled with 32P-dCTP by the random priming method by means of aPrimeTime kit (International Biotechnologies, Inc., New Haven, Conn.).Hybridization conditions are 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO4 pH 7.0, 1 mM EDTA at 50 C. After hybridization overnight, thefilters are washed with 2×SSC, 1% SDS. Positively hybridizing plaquesare detected by autoradiography. After purification to single plaques,cDNA inserts are isolated, and their sequences determined by the chaintermination method using dideoxy terminators labeled with fluorescentdyes (Applied Biosystems, Inc., Foster City, Calif.).

The standard experimental protocol described above can be used by one ofskill in the art to obtain protox genes sequentially homologous to theknown protox coding sequences from any other eukaryote, particularlyother higher plant species.

An alignment of the predicted amino acid sequences of the respectiveproteins encoded by the sequences shown in SEQ ID NOS: 2 and 6 are setforth in Table 1. An alignment of the predicted amino acid sequences ofthe respective proteins encoded by the sequences shown in SEQ ID NOS: 4and 8 are set forth in Table 2.

TABLE 1 Comparison of the Arabidopsis (SEQ ID No. 2) and Maize (SEQ IDNo. 6) Protox-1 Amino Acid Sequences Percent Similarity: 87.137 PercentIdentity: 78.008 Protox-1.Pep x Mzprotox-1.Pep  51GGTTITTDCVIVGGGISGLCIAQALATKHPDAAPNLIVTEAKDRVGGNII 100     ..|||:|||||||||.||||||:|  :..:::||||:.|.||||.   1....NSADCVVVGGGISGLCTAQALATRH..GVGDVLVTEARARPGGNIT 44 101T..REENGFLWEEGPNSFQPSDPMLTMVVDSGLKDDLVLGDPTAPRFVLW 148|  |.|:|:||||||||||||||:|||.||||||||||:|||.|||||||  45TVERPEEGYLWEEGPNSFQPSDPVLTMAVDSGLKDDLVFGDPNAPRFVLW 94 149NGKLRPVPSKLTDLPFFDLMSIGGKIRAGFGALGIRPSPPGREESVEEFV 198:||||||||| .||||||||||.||:|||:|||||||.||||||||||||  95EGKLRPVPSKPADLPFFDLMSIPGKLRAGLGALGIRPPPPGREESVEEFV 144 199RRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEQNGGSIIGG 248|||||.||||||||||||||||||||||||||||||||:||:.||||||| 145RRNLGAEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWRLEETGGSIIGG 194 249TFKAIQERKNAPKAERDPRLPKPQGQTVGSFRKGLRMLPEAISARLGSKV 298|:|.||||...||:.||:|||||.||||:|||||| |||:||...||||| 195TIKTIQERSKNPKPPRDARLPKPKGQTVASFRKGLAMLPNAITSSLGSKV 244 299KLSWKLSGITKLESGGYNLTYETPDGLVSVQSKSVVMTVPSHVASGLLRP 348||||||.:||| :. || |.||||:|:||||.|||:||:||.|||.:||| 245KLSWKLTSITKSDDKGYVLEYETPEGVVSVQAKSVIMTIPSYVASNILRP 294 349LSESAANALSKLYYPPVAAVSISYPKEAIRTECLIDGELKGFGQLHPRTQ 398||..||:|||::||||||||.:||||||||.||||||||.||||||||.| 295LSSDAADALSRFYYPPVAAVTVSYPKEAIRKECLIDGELQGFGQLHPRSQ 344 399GVETLGTIYSSSLFPNRAPPGRILLLNYIGGSTNTGILSKSEGELVEAVD 448|||||||||||||||||||.||:||||||||.|||||:||.|:||||||| 345GVETLGTIYSSSLFPNRAPDGRVLLLNYIGGATNTGIVSKTESELVEAVD 394 449RDLRKMLIKPNSTDPLKLGVRVWPQAIPQFLVGHFDILDTAKSSLTSSGY 498||||||||.....||| |||||||||||||||||:|:|:.||..|..:|| 395RDLRKMLINSTAVDPLVLGVRVWPQAIPQFLVGHLDLLEAAKAALDRGGY 444 499EGLFLGGNYVAGVALGRCVEGAYETAIEVNNFMSRYAYK* 538:|||||||||||||||||||||||.| ::.:|:.:||||| 445DGLFLGGNYVAGVALGRCVEGAYESASQISDFLTKYAYK* 484

Identical residues are denoted by the vertical bar between the twosequences. Alignment is performed using the GAP program described inDeveraux et al., Nucleic Acids Res. 12:387-395 (1984).

TABLE 2 Comparison of the Arabidopsis (SEQ ID No. 4) and Maize (SEQ IDNO. 8) Protox-2 Amino Acid Sequences Percent Similarity: 75.889 PercentIdentity: 57.905 Protox-2.Pep x Mzprotox-2.Pep   1............................MASGAVAD.HQIEAVSGKRVAV 21                            .|  |:|: .:  |..::.|||   1MLALTASASSASSHPYRHASAHTRRPRLRAVLAMAGSDDPRAAPARSVAV 50  22VGAGVSGLAAAYKLKSRGLNVTVFEADGRVGGKLRSVMQNGLIWDEGANT 71||||||||||||:|: .|:|||||||.:|.|||:|.  :.|::|||||||  51VGAGVSGLAAAYRLRQSGVNVTVFEAADRAGGKIRTNSEGGFVWDEGANT 100  72MTEAEPEVGSLLDDLGLREKQQFPISQKKRYIVRNGVPVMLPTNPIELVT 121|||:| |.:.|:|||||.:|||:| ||.|||||::|.|.::|.:||.|:. 101MTEGEWEASRLIDDLGLQDKQQYPNSQHKRYIVKDGAPALIPSDPISLMK 150 122SSVLSTQSKFQILLEPFLWKK....KSSKVSDASAEESVSEFFQRHFGQE 167||||||.||:.:::||||:||    .|:|||:.  .|||:.| :||||.| 151SSVLSTKSKIALFFEPFLYKKANTRNSGKVSEEHLSESVGSFCERHFGRE 200 168VVDYLIDPFVGGTSAADPDSLSMKHSFPDLWNVEKSFGSIIVGAIRTKFA 217||||::||||:||||:||:|||::|.||.|||:|:.:||:||||| .|:| 201VVDYFVDPFVAGTSAGDPESLSIRHAFPALWNLERKYGSVIVGAILSKLA 250 218AKGGKSRDTKSSPGTKKGSRGSFSFKGGMQILPDTLCKSLSHDEINLDSK 267|||:. :. ..|.|.::..|.||||.|||| | :.| ..::.|::.|:.. 251AKGDPVKTRHDSSGKRRNRRVSFSFHGGMQSLINALHNEVGDDNVKLGTE 300 268VLSLS..YNSGSRQENWSLSCVSHNETQRQ...NPHYDAVIMTAPLCNVK 312||||.  :::..  :.||:|. |.:..:::   |. :|||||||||:||: 301VLSLACTFDGVPALGRWSISVDSKDSGDKDLASNQTFDAVIMTAPLSNVR 350 313EMKVMKGGQPFQLNFLPEINYMPLSVLITTFTKEKVKRPLEGFGVLIPSK 362 ||. |||.|. |:|||.::|:|||:::|.|.|:.||:|||||||||| | 351RMKFTKGGAPVVLDFLPKMDYLPLSLMVTAFKKDDVKKPLEGFGVLIPYK 400 363E.QKHGFKTLGTLFSSMMFPDRSPSDVHLYTTFIGGSRNQELAKASTDEL 411| ||||:|||||||||||||||.|.| .|||||:|||:|.:|| |.|. | 401EQQKHGLKTLGTLFSSMMFPDRAPDDQYLYTTFVGGSHNRDLAGAPTSIL 450 412KQVVTSDLQRLLGVEGEPVSVNHYYWRKAFPLYDSSYDSVMEAIDKMEND 461||.|||||.:||||||:|. |.| || .|||||: .|.||:|||:|||.: 451KQLVTSDLKKLLGVEGQPTFVKHVYWGNAFPLYGHDYSSVLEAIEKMEKN 500 462LPGFFYAGNHRGGLSVGKSIASGCKAADLVISYLESCSNDKKPNDSL* 509||||||||| ::||.||. ||||:|||||.|||||| ......: 501LPGFFYAGNSKDGLAVGSVIASGSKAADLAISYLESHTKHNNSH*... 545

Example 4 Isolation of a Contaminating Yeast Protox Clone from anArabidopsis cDNA Library

In an effort to identify any rare cDNAs with protox activity, a secondscreen of the pFL61 Arabidopsis library was done as before, againyielding hundreds of complementing clones. Approximately 600 of thesewere patched individually onto gridded plates and incubated at 28° C.for 18 hours. Duplicate filter lifts were made onto Colony/Plaque screen(NEN) membranes according to the manufacturer's instructions. TheProtox-1 and Protox-2 cDNAs were removed from their vectors by digestionwith EcoRI/XhoI and by NotI, respectively. The inserts were separated bygel electrophoresis in 1.0% SeaPlaque GTG (FMC) agarose, excised, and³²P-labeled by random priming (Life Technologies). One set of lifts washybridized with each probe. Hybridization and wash conditions were asdescribed in Church and Gilbert, 1984.

Colonies (˜20) that failed to show clear hybridization to Protox-1 orProtox-2 were amplified in liquid culture and plasmid DNA was prepared.The DNA's were digested with NotI, duplicate samples were run on a 1.0%agarose gel, and then Southern blotted onto a Gene Screen Plus (NEN)filter. Probes of the two known Protox genes were labeled and hybridizedas before. There were two identical clones that were not Protox-1 orProtox-2. This clone was shown to recomplement the SASX38 mutant,although it grows very slowly, and was designated Protox-3.

Protox-3, in the pFL61 vector, was deposited Jun. 8, 1994 as pWDC-5(NRRL #B-21280). This coding sequence has been determined to be derivedfrom yeast DNA which was present as a minor contaminant in theArabidopsis cDNA library. The yeast DNA encoding protox-3 contained inpWDC-5 is set forth in SEQ ID NO:9 below.

Example 5 Demonstration of Plant Protox Clone Sensitivity to ProtoxInhibitory Herbicides in a Bacterial System.

Liquid cultures of Protox-1/SAS×38, Protox-2/SAS×38 andpBluescript/XL1-Blue were grown in L amp¹⁰⁰. One hundred microliteraliquots of each culture were plated on L amp¹⁰⁰ media containingvarious concentrations (1.0 nM-10 mM) of a protox inhibitory aryluracilherbicide of formula XVII. Duplicate sets of plates were incubated for18 hours at 37° C. in either low light or complete darkness.

The protox⁺ E. coli strain XL1-Blue showed no sensitivity to theherbicide at any concentration, consistent with reported resistance ofthe native bacterial enzyme to similar herbicides. The Protox-1/SAS×38was clearly sensitive, with the lawn of bacteria almost entirelyeliminated by inhibitor concentrations as low as 10 nM. TheProtox-2/SAS×38 was also sensitive, but only at a higher concentration(10 μM) of the herbicide. The effect of the herbicide on both plantprotox strains was most dramatic in low light, but was also apparent onplates maintained entirely in the dark. The toxicity of the herbicidewas entirely eliminated by the addition of 20mg/ml hematin to theplates.

The different herbicide tolerance between the two plant Protox strainsis likely the result of differential expression from these two plasmids,rather than any inherent difference in enzyme sensitivity.Protox-1/SAS×38 grows much more slowly than Protox-2/SAS×38 in anyheme-deficient media. In addition, the MzProtox-2/SAS×38 strain, with agrowth rate comparable to Arab Protox-1/SAS×38, is also very sensitiveto herbicide at the lower (10-100 nM) concentrations. Initialcharacterization of the yeast Protox-3 clone indicated that it also isherbicide sensitive.

Example 6 Selecting for Plant Protox Genes Resistant toProtox-inhibitory Herbicides in the E. coli Expression System.

Inhibition of plant protox enzymes in a bacterial system is useful forlarge-scale screening for herbicide-resistant mutations in the plantgenes. Initial dose response experiments, done by plating from liquidcultures, gave rise to high frequency “resistant” colonies even at highconcentrations of herbicide. This resistance was not plasrnid-bome,based on retransformation/herbicide sensitivity assay. TransformingProtox plasmids into the SAS×38 mutant and plating directly onto platescontaining herbicide reduces this background problem almost entirely.

The plant protox plasmids are mutagenized in a variety of ways, usingpublished procedures for chemical (e.g. sodium bisulfite (Shortle etal., Methods Enzymol. 100:457468 (1983); methoxylamine (Kadonaga et al.,Nucleic Acids Res. 13:1733-1745 (1985); oligonucleotide-directedsaturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA,83:710-714 (1986); or various polymerase misincorporation strategies(see, e.g. Shortle et al., Proc. Natl. Acad. Sci. USA, 79:1588-1592(1982); Shiraishi et al., Gene 64:313-319 (1988); and Leung et al.,Technique 1:11-15 (1989)). The expected up-promoter mutants fromwhole-plasmid mutagenesis are eliminated by recloning the codingsequence into a wild-type vector and retesting. Given that higherexpression is likely to lead to better growth in the absence ofherbicide, a visual screen for coding sequence mutants is also possible.

Any plant protox gene expressing herbicide resistance in the bacterialsystem may be engineered for optimal expression and transformed intoplants using standard techniques as described herein. The resultingplants may then be treated with herbicide to confirm and quantitate thelevel of resistance conferred by the introduced protox gene.

Example 7 Constructs for Expression of Herbicide-resistant MicrobialProtox Gene(s) in Plants.

The coding sequences for the B. subtilis protox gene hemY (Hansson andHederstedt, J. Bacteriol. 174: 8081 (1992); Dailey et al., J. Biol.Chem. 269: 813 (1994)) and for the E. coli protox gene hemG (Sasarman etal., Can. J. Microbiol. 39: 1155 (1993)) were isolated from laboratorystrains by PCR amplification using standard conditions and flankingprimers designed from the published sequences. These genes are known tocode for herbicide-resistant forms of the protox enzyme.

Using standard techniques of overlapping PCR fusion (Ausubel et al.,Current Protocols in Molecular Biology. John Wiley & Sons, Inc. (1994)),both bacterial genes were fused to two different Arabidopsis chloroplasttransit peptide sequences (CTPs). The first was the CTP from theacetohydroxy acid synthase (AHAS, Mazur et al., Plant Physiol. 85: 1110(1987)), which should allow import into the stroma of the chloroplast.The second was from the Arabidopsis plastocyanin gene (Vorst et al.,Gene 65: 59 (1988)), which has a bipartite transit peptide. The aminoterminal portion of this CTP targets the protein into the chloroplast,where the carboxy terminus routes it into the thylakoid membranes. Allfour gene fusions were cloned behind the 2×35S promoter in a binaryexpression vector designed for production of transgenic plants byagrobacterium transformation.

Following isolation of the Arabidopsis and maize protox cDNAs, thechloroplast transit peptide from Protox-1 or MzProtox-1 may also befused to the two bacterial protox proteins in the same manner as above.

The vectors described above may then be transformed into the desiredplant species and the resulting transforrnants assayed for increasedresistance to herbicide.

Example 8 Domain Switching Between Arabidopsis/B. subtilis Genes toProduce Chimeric, Herbicide Resistant Protox.

One approach that may be used to generate a protox gene which is bothherbicide resistant and capable of providing effective protox enzymaticactivity in a plant is to fuse portion(s) of a bacterial and plantprotox gene. The resulting chimeric genes may then be screened for thosewhich are capable of providing herbicide resistant protox activity in aplant cell. For instance, the Arabidopsis and the B. subtilis (hemY)protox peptide sequences are reasonably colinear with regions of highhomology. The hemY coding sequence is cloned into pBluescript and testedfor its ability to express herbicide-resistant protox activity inSASX38. Protox-1/hemY chimeric genes are constructed using fusion PCRtechniques, followed by ligation back into the pBluescript vector. Theinitial exchange is approximately in the middle of the proteins. Thesefusions are tested for protox function by complementation, and thenassayed for herbicide resistance by plating on herbicide with intactProtox-1 and hemY controls.

Example 9 Production of Herbicide-tolerant Plants by Overexpression ofPlant Protox Genes.

To express the Arabidopsis or maize protein in transgenic plants, theappropriate full length cDNA was inserted into the plant expressionvector pCGN1761ENX, which was derived from pCGN1761 as follows. pCGN1761was digested at its unique EcoRI site, and ligated to a double-strandedDNA fragment comprised of two oligonucleotides of sequence 5′ AAT TATGAC GTA ACG TAG GAA TTA GCG GCCC GCT CTC GAG T 3′ (SEQ ID NO: 11) and 5′AAT TAC TCG AGA GCG GCC GCG AAT TCC TAC GTT ACG TCA T 3′ (SEQ ID NO:12). The resulting plasmid, pCGN1761ENX, contained unique EcoRI, NotI,and XhoI sites that lie between a duplicated 35S promoter fromcauliflower mosaic virus (Kay et al., Science 236:1299-1302 (1987)) andthe 3′ untranslated sequences of the tml gene of Agrobacteriumtumefaciens. This plasmid is digested and ligated to a fragmentresulting from restriction enzyme digestion of one of the plasmidsbearing a protox cDNA, such that it carries the complete protox cDNA.From this plasmid is excised an XbaI fragment comprising the Arabidopsisprotox cDNA flanked by a duplicated 35S promoter and the 3′ untranslatedsequences of the tml gene of A. tumefaciens. This XbaI fragment isinserted into the binary vector pCIB200 at its unique XbaI site, whichlies between T-DNA border sequences. The resulting plasmid, designatedpCIB200protox, is transformed into A. tumefaciens strain CIB542. See,e.g. Uknes et al., Plant Cell 5:159-169 (1993).

Leaf disks of Nicotiana tabacum cv. Xanthi-nc are infected with A.tumefaciens CEB542 harboring pCIB200IGPD as described by Horsch et al,Science 227: 1229 (1985). Kanamycin-resistant shoots from 15 independentleaf disks are transferred to rooting medium, then transplanted to soiland the resulting plants grown to maturity in the greenhouse. Seed fromthese plants are collected and germinated on MS agar medium containingkanamycin. Multiple individual kanarnycin resistant seedlings from eachindependent primary transfornant are grown to maturity in thegreenhouse, and their seed collected. These seeds are germinated on MSagar medium containing kanamycin.

Plant lines that give rise to exclusively kanamycin resistant seedlingsare homozygous for the inserted gene and are subjected to furtheranalysis. Leaf disks of each of the 15 independent transgenic lines areexcised with a paper punch and placed onto MS agar containing variousincreasing concentrations of a protox inhibitory herbicide.

After three weeks, two sets of 10 disks from each line were weighed, andthe results recorded. Transgenic lines more resistant to the inhibitorthan wild type, non-transformed plants are selected for furtheranalysis.

RNA is extracted from leaves of each of these lines. Total RNA from eachindependent homozygous line, and from non-transgenic control plants, isseparated by agarose gel electrophoresis in the presence of formaldehyde(Ausubel et al., Current Protocols in Molecular Biology, Wiley & Sons,New York (1987)). The gel is blotted to nylon membrane (Ausubel et al.,supra.) and hybridized with the radiolabeled Arabidopsis protox cDNA.Hybridization and washing conditions are as described by Church andGilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984). The filter isautoradiographed, and intense RNA bands corresponding to the protoxtransgene are detected in all herbicide-tolerant transgenic plant lines.

To further evaluate resistance of the protox-overexpressing line, plantsare grown in the greenhouse and treated with various concentrations of aprotox-inhibiting herbicide.

Example 10 Growth of Tobacco Cell Suspension Cultures

Media:

MX 1: This medium consists of Murashige and Skoog (“MS”, T. Murashige etal., Physiol. Plant. 15:473497, 1962) major salts, minor salts andFe-EDTA (Gibco # 500-1117; 4.3 g/l), 100 mg/l myo-inositol, 1 mg/lnicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamine -HCl, 2-3 g/lsucrose, 0.4 mg/l 2,4-dichlorophenoxyacetic acid, and 0.04 mg/l kinetin,pH 5.8. The medium is sterilized by autoclaving.

N6: This medium comprises macroelements, microelements and Fe-EDTA asdescribed by C -C. Chu et al., Scientia Sinica 18:659 (1975), and thefollowing organic compounds: Pyridoxine-HCl (0.5 mg/l), thiamine-HCl(0.1 mg/l), nicotinic acid (0.5 mg/l), glycine (2.0 mg/l), and sucrose(30.0 g/l). The solution is autoclaved. The final pH is 5.6.

Remarks: Macroelements are made up as a 10× concentrated stock solution,and microelements as a 1000× concentrated stock solution. Vitamin stocksolution is normally prepared 100× concentrated.

Suspension cultured cells of Nicotiana tabacum, line S3, are grown inliquid culture medium Mx1. 100 ml Erlenmeyer flasks containing 25 mlmedium MX1 are inoculated with 10 ml of a cell culture previously grownfor 7 days. Cells are incubated at 25° C. in the dark on an orbitalshaker at 100 rpm (2 cm throw). Cells are subcultured at 7 day intervalsby inoculating an aliquot sample into fresh medium, by decanting orpipetting off around 90% of the cell suspension followed by replenishingfresh medium to give the desired volume of suspension. 5-8 grams offresh weight cell mass are produced within 10 days of growth from aninoculum of 250-350 mg cells.

Example 11 Production of Tobacco Cell Cultures Tolerant to HerbicidalProtox Inhibitors by Plating Cells on Solidified Selection Medium

Cells are pregrown as in Example 10. Cells are harvested by allowingcells to sediment, or by brief centrifugation at 500×g, and the spentculture medium is removed. Cells are then diluted with fresh culturemedium to give a cell density suitable for cell plating, about 10,000colony forming units per ml. For plating, cells in a small volume ofmedium (approx. 1 ml) are evenly spread on top of solidified culturemedium (MX1, 0.8% agar) containing the desired concentration of theinhibitor. About 20-30 ml of medium are used per 10 cm Petri plate. Thesuitable inhibitor concentration is determined from a dose-responsecurve (Example 14), and is at least twofold higher than the IC₅₀ ofsensitive wild-type cells.

Culture plates containing cells spread onto selection medium areincubated under normal growth conditions at 25-28° C. in the dark untilcell colonies are formed. Emerging cell colonies are transferred tofresh medium containing the inhibitor in the desired concentration.

In a preferred modification of the described method the pregrownsuspension of cultured cells is first spread in a small volume of liquidmedium on top of the solidified medium. An equal amount of warm liquidagar medium (1.2-1.6% agar) kept molten at around 40° C. is added andthe plate gently but immediately swirled to spread the cells evenly overthe medium surface and to mix cells and agar medium, before the mediumsolidifies.

Alternatively, the cells are mixed with the molten agar medium prior tospreading on top of the selection medium. This method has the advantagethat the cells are embedded and immobilized in a thin layer ofsolidified medium on top of the selection medium. It allows for betteraeration of the cells as compared to embedding cells in the whole volumeof 20-30 ml.

Example 12 Production of Tobacco Cell Cultures Tolerant to a HerbicidalProtox Inhibitor by Growing Cells in Liquid Selection Medium

Cells cultured as in Example 10 are inoculated at a suitable celldensity into liquid medium MX1 containing the desired concentration of aherbicidal protox inhibitor. Cells are incubated and grown as in Example10. Cells are subcultured, as appropriate depending on the rate ofgrowth, using fresh medium containing the desired inhibitorconcentration after a period of 7-10 days.

Depending on the inhibitor concentration used, cell growth may be slowerthan in the absence of inhibitor.

Example 13 Production of Tobacco Cells with Enhanced Levels of ProtoxEnzyme

In order to obtain cell cultures or callus with enhanced levels ofprotox enzyme, suspension cultures or callus are transferred, in astep-wise manner, to increasingly higher concentrations of herbicidalprotox inhibitor. In particular, the following steps are performed:

Cell colonies emerging from plated cells of Example 11 are transferredto liquid MX1 medium containing the same concentration of protoxinhibitor as used in the selection according to Example 11 in order toform suspension cultures. Alternatively, selected cell suspensioncultures of Example 12 are subcultured in liquid MX1 medium containingthe same concentration of protox inhibitor as used for selectionaccording to Example 12.

Cultures are subcultured 1-20 times at weekly intervals and are thensubcultured into MX1 medium containing the next higher herbicideconcentration. The cells are cultured for 1-10 subcultures in mediumcontaining this higher concentration of herbicide. The cells are thentransferred to MX1 medium containing the next higher concentration ofherbicide.

Alternatively, pieces of selected callus of Example 11 are transferredto solidified MX1 medium supplemented with the desired herbicideconcentration. Transfer to higher herbicide concentrations follows theprocedure outlined in the preceding paragraph except that solidifiedmedium is used.

Example 14 Measuring Herbicide Dose-dependent Growth of Cells inSuspension Cultures

In order to obtain a dose-response curve the growth of cells atdifferent concentrations of herbicide is determined. Suspension culturecells of herbicidal protox inhibitor sensitive wild-type tobacco cellsS3 and herbicide tolerant selected or transgenic cells S3 and herbicidetolerant selected or transgenic cells are pregrown in liquid medium asin Example 11 at a high cell density for 2-4 days. The cells are washedfree of spent medium and fresh medium without herbicide is added to givethe desired cell density (about 150 mg FW cells per ml of suspension). Asample of 2.5 ml of cell suspension, containing approx. 250-300 mg FWcells, is then inoculated into approx. 30 ml of liquid medium of desiredherbicide concentration contained in a 100 ml Erlenmeyer flask. Care istaken to inoculate the same amount of cells into each flask. Each flaskcontains an equal volume of medium. 3-6 replicate flasks are inoculatedper herbicide concentration. The herbicide concentration is selectedfrom zero (=control), 0.1 ppb, 0.3 ppb, 1 ppb, 3 ppb, 10 ppb, 30 ppb,100 ppb, 300 ppb, 1000 ppb, 3000 ppb, and 10,000 ppb. Several samples ofinoculated cells are also taken at the time of inoculation to determinethe mass of cells inoculated per flask.

Cells are then incubated for growth under controlled conditions at 28°in the dark for 10 days. The cells are harvested by pouring the contentsof each flask onto a filter paper disk attached to a vacuum suctiondevice to remove all liquid and to obtain a mass of reasonably dry freshcells. The fresh mass of cells is weighed. The dry weight of samples maybe obtained after drying.

Cell growth is determined and expressed as cell gain within 10 days andexpressed as a percentage relative to cells grown in the absence ofherbicide according to the formula: (final mass of herbicide-grown cellsminus inoculum mass ×100 divided by final mass of cells grown withoutherbicide minus inoculum mass). IC₅₀ values are determined from graphsof plotted data (relative cell mass vs. herbicide concentration). IC₅₀denotes the herbicide concentration at which cell growth is 50% ofcontrol growth (cells grown in the absence of herbicide).

In a modification of the method several pieces of callus derived from aherbicide resistant cell culture, as obtained in Examples 11 and 13, aretransferred to solidified callus culture medium containing the differentherbicide concentrations. Relative growth is determined after a culture.period of 2-6 weeks be weighing callus pieces and comparing to a controlculture grown in medium without herbicide. However, the suspensionmethod is preferred for its greater accuracy.

Example 15 Determination of Cross Tolerance

In order to determine the extent at which cells show tolerance toanalogous or other herbicides, Example 14 is repeated by growing cellsin increasing concentrations of chosen herbicides. The relative growthof the cells and their IC₅₀ value is determined for each herbicide forcomparison.

Example 16 Determining the Stability of the Herbicide TolerancePhenotype Over Time

In order to determine whether the herbicide tolerant phenotype of a cellculture is maintained over time, cells are transferred from herbicidecontaining medium to medium without herbicide. Cells are grown, asdescribed in Example 10, in the absence of herbicide for a period of 3months, employing regular subculturing at suitable intervals (7-10 daysfor suspension cultures; 3-6 weeks for callus cultures). A knownquantity of cells is then transferred back to herbicide containingmedium and cultured for 10 days (suspension cultures) or 4 weeks (calluscultures). Relative growth is determined as in Example 14.

Example 17 Induction and Culture of Embryogenic Callus from CornScutellum Tissue

Ears are harvested from self pollinated corn plants of the inbred lineFunk 2717 12-14 days post pollination. Husks are removed and the earsare sterilized for about 15 minutes by shaking in a 20% solution ofcommercial Chlorox bleach with some drops of detergent added for betterwetting. Ears are then rinsed several times with sterile water. Allfurther steps are performed aseptically in a sterile air flow hood.Embryos of 1.5-2.5 mm length are removed from the kernels with a spatulaand placed, embryo axis downwards, onto MS culture medium containing 2mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3% sucrose, solidifiedwith 0.24% Gelrite^(R).

Embryogenic callus forms on the scutellum tissue of the embryos within24 weeks of culture at about 28° C. in the dark. The callus is removedfrom the explant and transferred to fresh solidified MS mediumcontaining 2 mg/l 2,4-D. The subculture of embryogenic callus isrepeated at weekly intervals. Only callus portions having an embryogenicmorphology are subcultured.

Example 18 Selection of Corn Cell Cultures Tolerant to Herbicidal ProtoxInhibitors

a) Selection using embrogenic callus:

Embryogenic callus of Example 17 is transferred to callus maintenancemedium consisting of N6 medium containing 2 mg/l 2,4-D, 3% sucrose andprotox inhibitor at a concentration sufficient to retard growth, butthat does not affect the embyrogenicity of the culture, and solidifiedwith 0.24% Gelrite^(R). To increase the frequency of herbicide tolerantmutations, cultures can be pretreated before selection with a chemicalmutagen, e.g. ethylmethane sulfonate, or a physical mutagen, e.g. UVlight, at a concentration just below the concentration at which growthinhibition is detected, as determined in Example 14. Cultures areincubated at 28° C. in the dark. After 14 days growing callus istransferred to fresh medium of the same composition. Only cultures withthe desired embryogenic morphology known as friable embryogenic callusof type II morphology are subcultured. Cultures are propagated bysubculturing at weekly intervals for two to ten subcultures on freshmedium whereby only the fastest growing cultures are subcultured. Thefast growing callus is then transferred to callus maintenance mediumcontaining a protox inhibiting herbicide at a suitable concentration asdefined in Example 11. When callus grows well on this herbicideconcentration, usually after about five to ten weekly subcultures, thecallus is transferred to callus maintenance medium containing athree-fold higher concentration of inhibitor, and subcultured until awell growing culture is obtained. This process is repeated using mediumcontaining protox inhibitor at a concentration 10-fold higher than theoriginal suitable concentration, and again with medium containing20-fold and 40-fold higher concentrations.

When sufficient callus has been produced it is transferred toregeneration medium suitable for embryo maturation and plantregeneration. Embryogenic callus growing on each of the herbicideconcentrations used is transferred to regeneration medium.

b) Selection usina embryogenic suspension cultures:

Embryogenic suspension cultures of corn Funk inbred line 2717 areestablished according to Example 24 and maintained by subculturing atweekly intervals to fresh liquid N6 medium containing 2 mg/l 2,4-D. Toincrease the frequency of herbicide tolerant mutations, cultures can betreated at this time with a chemical mutagen, e.g. ethylmethanesulfonate, at a concentration just below the concentration at whichgrowth inhibition is detected, as determined in Example 14. Forselection, the cultures are transferred to liquid N6 medium containing 2mg/l 2,4-D and a concentration of inhibitor sufficient to retard growth,but that does not affect the embyrogenicity of the culture. Cultures aregrown on a shaker at 120 rpm at 28° C. in the dark. At weekly intervals,the medium is removed and fresh medium added. The cultures are dilutedwith culture medium in accord with their growth to maintain about 10 mlof packed cell volume per 50 ml of medium. At each subculture, culturesare inspected and only fast growing cultures with the desired friableembryogenic morphology are retained for further subculture. After two toten subcultures in N6 medium containing, cultures are increasing ingrowth rate at least two- to threefold per weekly subculture. Thecultures are then transferred to N6 medium containing 2 mg/l 2,4-D and athree-fold higher dose of inhibitor than originally used. Growingcultures are repeatedly subcultured in this medium for another two toten subcultures as described above. Fast growing cultures with thedesired friable embryogenic morphology are selected for furthersubculture. Fast growing cultures are then transferred to N6 mediumcontaining 2 mg 2,4-D and a ten-fold higher concentration of inhibitorthan originally used, and the process of subculturing growing cultureswith the desired friable embryogenic morphology is repeated for two toten subcultures until fast growing cultures are obtained. These culturesare then transferred to N6 medium containing 2 mg/l 2,4-D and a 30-foldhigher concentration of inhibitor than originally used.

For regeneration of plants from each embryogenic suspension cultureselected with the mentioned herbicide concentration level, the culturesare first transferred onto N6 medium solidified with 0.24% Gelrite^(R)and containing 2 mg/l 2,4-D and, optionally, the concentration ofinhibitor in which the cultures have been growing, to produceembryogenic callus. The embryogenic callus is subcultured onto freshcallus maintenance medium until a sufficient amount of callus isobtained for regeneration. Only cultures with the desired embryogenicmorphology are subcultured.

Example 19 Regeneration of Corn Plants form Selected Callus orSuspension Culture

Plants are regenerated from the selected embryogenic callus cultures ofExample 13 by transferring to fresh regeneration medium. Regenerationmedia used are: 0N6 medium consisting of N6 medium lacking 2,4-3, or N61consisting of N6 medium containing 0.25 mg/l 2,4-D and 10 mg/l kinetin(6-furfurylaminopurine), or N62 consisting of N6 medium containing 0.1mg/l 2,4-D and 1 mg/l kinetin, all solidified with 0.24% Gelrite^(R).Cultures are grown at 28° C. in the light (16 h per day of 10-100μEinsteins/m²sec from white fluorescent lamps). The cultures aresubcultured every two weeks onto fresh medium. Plantlets develop within3 to 8 weeks. Plantlets at least 2 cm tall are removed from adheringcallus and transferred to root promoting medium. Different rootpromoting media are used. The media consist of N6 or MS medium lackingvitamins with either the usual amount of salts or with salts reduced toone half, sucrose reduced to 1 g/l, and further either lacking growthregulating compounds or containing 0.1 mg/l a-naphthaleneacetic acid.Once roots are sufficiently developed, plantlets are transplanted to apotting mixture consisting of vermiculite, peat moss and garden soil. Attransplanting all remaining callus is trimmed away, all agar is rinsedoff and the leaves are clipped about half. Plantlets are grown in thegreenhouse initially covered for some days with an inverted clearplastic cup to retain humidity and grown with shading. Afteracclimatization plants are repotted and grown to maturity. FertilizerPeters 20-20-20 is used to ensure healthy plant development. Uponflowering plants are pollinated, preferably self pollinated.

Example 20 Construction of Plant Transformation Vectors

Numerous transformation vectors are available for plant transformation,and the genes of this invention can be used in conjunction with any suchvectors. The selection of vector for use will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene whichconfers resistance to the herbicide phosphinothricin (White et al., NuclAcids Res 18: 1062 (1990), Spencer et al. Theor Appl Genet 79:625-631(1990)), the hph gene which confers resistance to the antibiotichygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), andthe dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J. 2(7): 1099-1104 (1983)).

(1) Construction of Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) andpXYZ. Below the construction of two typical vectors is described.

Construction of pCIB200 and pCIB2001

The binary vectors pCIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and was constructed inthe following manner. pTJS75kan was created by NarI digestion of pTJS75(Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride etal., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers wereligated to the EcoRV fragment of pCIB7 which contains the left and rightT-DNA borders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153-161 (1987)), and theXhoI-digested fragment was cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19[1338]). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, Sstl, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200 which created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, Sall, MluI, BclI, AvrII, ApaI, HpaI, and StuI. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

Construction of pCIB10 and Hygromycin Selection Derivatives Thereof

The binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants, T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al., Gene 53: 153-161 (1987). Variousderivatives of pCIB10 have been constructed which incorporate the genefor hygromycin B phosphotransferase described by Gritz et al., Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCB715,pCIB717). ps (2) Construction of Vectors Suitable for non-AgrobacteriumTransformation.

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques which do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Below, the construction of some typical vectors isdescribed.

Construction of pCIB3064

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites were mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites were 96and 37 bp away from the unique SalI site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 was designatedpCIB3025. The GUS gene was then excised from pCIB3025 by digestion withSalI and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 was obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes was excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). Thisgenerated pCIB3064 which comprises the bar gene under the control of theCAMV 35S promoter and terminator for herbicide selection, a gene froampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

Construction of pSOG19 and pSOG35

pSOG35 is a transformation vector which utilizes the E. coli genedihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR was used to amplify the 35S promoter(˜800 bp), intron 6 from the maize Adhl gene (˜550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250 bp fragment encodingthe E. coli dihydrofolate reductase type II gene was also amplified byPCR and these two PCR fragments were assembled with a SacI-PstI fragmentfrom pBI221 (Clontech) which comprised the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatedpSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generated the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign sequences.

Example 20 Construction of Plant Expression Cassettes

Gene sequences intended for expression in transgenic plants are firstlyassembled in expression cassettes behind a suitable promoter andupstream of a suitable transcription terminator. These expressioncassettes can then be easily transferred to the plant transformationvectors described above in Example 19.

Promoter Selection

The selection of a promoter used in expression cassettes will determinethe spatial and temporal expression pattern of the transgene in thetransgenic plant. Selected promoters will express transgenes in specificcell types (such as leaf epidermal cells, mesophyll cells, root cortexcells) or in specific tissues or organs (roots, leaves or flowers, forexample) and this selection will reflect the desired location ofexpression of the transgene. Alternatively, the selected promoter maydrive expression of the gene under a light-induced or other temporallyregulated promoter. A further alternative is that the selected promoterbe chemically regulated. This would provide the possibility of inducingexpression of the transgene only when desired and caused by treatmentwith a chemical inducer.

Transcriptional Terminators

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants and include the CAMV 35S terminator, the tmlterminator, the nopaline synthase terminator, the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.

Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adh1 gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronzel gene had a similar effect in enhancing expression (Callis etal., supra). Intron sequences have been routinely incorporated intoplant transformation vectors, typically within the non-translatedleader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990))

Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. For example, the targeting ofgene products to the chloroplast is controlled by a signal sequencefound at the amino terminal end of various proteins and which is cleavedduring chloroplast import yielding the mature protein (e.g. Comai et al.J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can befused to heterologous gene products to effect the import of heterologousproducts into the chloroplast (van den Broeck et al. Nature 313: 358-363(1985)). DNA encoding for appropriate signal sequences can be isolatedfrom the 5′ end of the cDNAs encoding the RUBISCO protein, the CABprotein, the EPSP synthase enzyme, the GS2 protein and many otherproteins which are known to be chloroplast localized.

Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms forrnitochondria. Targeting to cellular protein bodies has been describedby Rogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

In addition sequences have been characterized which cause the targetingof gene products to other cell compartments. Amino terminal sequencesare responsible for targeting to the ER, the apoplast, and extracellularsecretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783(1990)). Additionally, amino terminal sequences in conjunction withcarboxy terminal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al., Plant Molec. Biol. 14: 357-368 (1990)).

By the fusion of the appropriate targeting sequences described above totransgene sequences of interest it is possible to direct the transgeneproduct to any organelle or cell compartment. For chloroplast targeting,for example, the chloroplast signal sequence from the RUBISCO gene, theCAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame tothe amino terminal ATG of the transgene. The signal sequence selectedshould include the known cleavage site and the fusion constructed shouldtake into account any amino acids after the cleavage site which arerequired for cleavage. In some cases this requirement may be fulfilledby the addition of a small number of amino acids between the cleavagesite and the transgene ATG or alternatively replacement of some aminoacids within the transgene sequence. Fusions constructed for chloroplastimport can be tested for efficacy of chloroplast uptake by in vitrotranslation of in vitro transcribed constructions followed by in vitrochloroplast uptake using techniques described by (Bartlett et al. In:Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier. pp 1081-1091(1982); Wasmann etal. Mol. Gen. Genet. 205:446-453(1986)). These construction techniques are well known in the art and areequally applicable to mitochondria and peroxisomes. The choice oftargeting which may be required for expression of the transgenes willdepend on the cellular localization of the precursor required as thestarting point for a given pathway. This will usually be cytosolic orchloroplastic, although it may is some cases be mitochondrial orperoxisomal. The products of transgene expression will not normallyrequire targeting to the ER, the apoplast or the vacuole.

The above described mechanisms for cellular targeting can be utilizednot only in conjunction with their cognate promoters, but also inconjunction with heterologous promoters so as to effect a specific celltargeting goal under the transcriptional regulation of a promoter whichhas an expression pattern different to that of the promoter from whichthe targeting signal derives.

Example 21 Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques which do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species which are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432(tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No.4,795,855 (poplar)). Agrobacterium transformation typically involves thetransfer of the binary vector carrying the foreign DNA of interest (e.g.pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which maydepend of the complement of vir genes carried by the host Agrobacteriumstrain either on a co-resident Ti plasmid or chromosomally (e.g. strainCIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169(1993)). The transfer of the recombinant binary vector to Agrobacteriumis accomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877(1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Example 22 Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complex vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

Patent Applications EP 0 292 435 (to Ciba-Geigy), EP 0 392 225 (toCiba-Geigy) and WO 93/07278 (to Ciba-Geigy) describe techniques for thepreparation of callus and protoplasts from an {acute over (e)}liteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al., Plant Cell 2: 603-618 (1990)) and Frommet al., Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al.,Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of {acute over (e)}lite inbred lines of maize by particlebombardment. This technique utilizes immature maize embryos of 1.5-2.5mm length excised from a maize ear 14-15 days after pollination and aPDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques forthe generation, transformation and regeneration of Pooideae protoplasts.These techniques allow the transformation of Dactylis and wheat.Furthermore, wheat transformation was been described by Vasil et al.,Biotechnology 10: 667-674 (1992)) using particle bombardment into cellsof type C long-term regenerable callus, and also by Vasil et al.,Biotechnology 11: 1553-1558 (1993)) and Weeks et al., Plant Physiol.102: 1077-1084 (1993) using particle bombardment of immature embryos andimmature embryo-derived callus. A preferred technique for wheattransformation, however, involves the transformation of wheat byparticle bombardment of immature embryos and includes either a highsucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashige & Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos whichis allowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e. induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical. An appropriate gene-carryingplasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sizegold particles using standard procedures. Each plate of embryos is shotwith the DuPont Biolistics, helium device using a burst pressure of˜1000 psi using a standard 80 mesh screen. After bombardment, theembryos are placed back into the dark to recover for about 24 h (stillon osmoticum). After 24 hrs, the embryos are removed from the osmoticumand placed back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing theappropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2mg/l methotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contained half-strength MS, 2% sucrose, and the sameconcentration of selection agent. Patent application Ser. No. 08/147,161describes methods for wheat transformation and is hereby incorporated byreference.

Example 23 Selecting for Plant Protox Genes Resistant toProtox-inhibitory Herbicides in the E. coli Expression System

The plasmid pWDC-4, encoding the maize chloroplastic protox enzyme, istransformed into the random mutagenesis strain XL1-Red (Stratagene, LaJolla, Calif.). The transformation is plated on L media containing 50μg/ml ampicillin and incubated for 48 hours at 37° C. Lawns oftransformed cells are scraped from the plates and plasmid DNA preparedusing the Wizard Megaprep kit (Promega, Madison, Wis.). Plasmid DNAisolated from this mutator strain is predicted to contain approximatelyone random base change per 2000 nucleotides (see Greener et al.,Strategies 7(2):32-34 (1994)).

The mutated plasmid DNA is transformed into the hemG mutant SAS×38(Sasarman et al., J. Gen. Microbiol. 113: 297 (1979) and plated on Lmedia containing 100 μg/ml ampicillin and on the same media containingvarious concentrations of protox-inhibiting herbicide. The plates areincubated for 2-3 days at 37° C. Plasmid DNA is isolated from allcolonies that grow in the presence of herbicide concentrations thateffectively kill the wild type strain. The isolated DNA is thentransformed into SAS×38 and plated again on herbicide to ensure that theresistance is plasmid-borne.

Mutated pWDC-4 plasmid DNA is again isolated from resistant colonies andthe protox coding sequence is excised by digestion with EcoRI and XhoI.The excised protox coding sequence is then recloned into anunmutagenized pBluescript vector and retested for resistance toprotox-inhibiting herbicide in the same manner described above.

This process eliminates non-coding sequence mutations which conferresistance such as up-promoter mutants (i.e. mutants whose resistance isdue to mutations causing increased expression of unmodified protox) andleaves only mutants whose resistance is due to mutations in the protoxcoding sequence. The DNA sequence for all putative herbicide-tolerantprotox genes identified through this process is determined and mutationsare identified by comparison with the wild-type pWDC-4 protox sequence.

Using the procedure described above, a resistance mutation converting aC to a T at nucleotide 498 in the pWDC-4 sequence (SEQ ID No. 5) hasbeen identified. The plasmid carrying this mutation has been designatedpMzC-1Val. This change converts a GCT codon for alanine at amino acid166 (SEQ ID No. 6) to a GTT codon for valine and results in a protoxenzyme that is at least 10× more resistant to protox-inhibitingherbicide in the bacterial assay.

pMzC-1Val, in the pBluescript SK vector, was deposited on Sep. 30, 1994under the designation pWDC-8 with the Agricultural Research CultureCollection and given the deposit designation NRRL #21340.

The same strategy was used to screen for herbicide-resistant forms ofthe Arabidopsis Protox-1 gene in various vectors. One resistancemutation identified is a C to T change at nucleotide 689 in the pWDC-2sequence (SEQ ID No. 1); this plasmid is designated pAraC-1Val. Thischange is identical to the pMzC-1Val mutant above, converting a GCTcodon for alanine at amino acid 220 (SEQ ID No. 2) to a GTT codon forvaline at the corresponding position in the Arabidopsis protox proteinsequence.

A second resistant gene contains an A to G change at nucleotide 1307 inthe pWDC-2 sequence (SEQ ID No. 1); this plasmid is designatedpAraC-2Cys. This change converts TAC codon for tyrosine at amino acid426 (SEQ ID No. 2) to a TGC codon for cysteine. The correspondingtyrosine codon in the maize protox-1 sequence at nucleotide position1115-1117 (SEQ ID NO. 5; amino acid position 372 of SEQ ID NO. 6) may besimilarly mutated to generate a herbicide resistant form of this enzyme.

A third resistant mutant has a G to A change at nucleotide 691 in thepWDC-2 sequence (SEQ ID No. 1); this plasmid is designated pAraC-3Ser.This mutation converts GGT codon for glycine at amino acid 221 SEQ IDNo. 2) to an AGT codon for serine at the codon position adjacent to themutation in pAraC-1. The corresponding glycine codon in the maizeprotox-1 sequence at nucleotide position 497-499 (SEQ ID NO. 5; aminoacid position 167 of SEQ ID NO. 6) may be similarly mutated to generatea herbicide resistant form of this enzyme.

All the mutations described above result in a protox enzyme that is atleast 10× more resistant to protox-inhibiting herbicide in the bacterialassay.

pAraC-2Cys, in the pFL61 vector, was deposited on Nov. 14, 1994 underthe designation pWDC-7 with the Agricultural Research Culture Collectionand given the deposit designation NRRL #21339N.

A. Additional Herbicide-resistant Codon Substitutions at PositionsIdentified in the Random Screen

The amino acids identified as herbicide resistance sites in the randomscreen are replaced by other amino acids and tested for function and forherbicide tolerance in the bacterial system. Oligonucleotide-directedmutagenesis of the Arabidopsis Protox-1 sequence is performed using theTransformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, Calif.).After amino acid changes are confirmed by sequence analysis, the mutatedplasmids are transformed into SAS×38 and plated on L-amp¹⁰⁰ media totest for function and on various concentrations of protox-inhibitingherbicide to test for tolerance.

This procedure was applied to the alanine codon at nucleotides 688-690and to the tyrosine codon at nucleotides 1306-1308 of the Arabidopsisprotox sequence (SEQ ID No. 1). The results demonstrate that the alaninecodon at nucleotides 688-690 can be changed to a codon for valine,threonine, leucine or cysteine to yield a herbicide-resistant protoxenzyme which retains function. The results further demonstrate that thetyrosine codon at nucleotides 1306-1308 can be changed to a codon forcysteine, isoleucine, leucine, threonine or valine to yield aherbicide-resistant protox enzyme which retains function.

B. Demonstration of Resistant Mutations' Cross-tolerance to VariousProtox-inhibiting Compounds.

Resistant mutant plasmids, selected for resistance against a singleherbicide, are tested against a spectrum of other protox-inhibitingcompounds. The SAS×38 strain containing the wild-type plasmid is platedon a range of concentrations of each compound to determine the lethalconcentration for each one. Resistant mutant plasmids in SAS×38 areplated and scored for the ability to survive on a concentration of eachcompound which is at least 10 fold higher than the concentration that islethal to the SAS×38 strain containing the wild-type plasmid.

Results from cross-tolerance testing show that each of the mutationsidentified confer tolerance to a variety of protox inhibiting compounds.In particular, the results show that 1) the AraC1-Val mutation confersresistance to protox inhibitors including, but not necessarily limitedto, those having the Formulae IV, XI, XIII, XIV, XV and XVII; 2) theAraC-2Cys mutation confers resistance to protox inhibitors including,but not necessarily limited to, those having Formulae XI, XIII, XV andXVII; 3) the MzC-1Val mutation confers resistance to protox inhibitorsincluding, but not necessarily limited to, those having the Formulae XI,XII, XIII, XIV, XV, XVI and XVII; 4) the AraC-3Ser mutation confersresistance to protox inhibitors including, but not necessarily limitedto, bifenox and those having the Formulae IV, XII, XIII, XIV, XV, andXVII.

C. Production of Herbicide Tolerant Plants by Overexpression of PlantProtox Genes

The Arabidopsis Protox-l coding sequences from both the wild-type andthe resistant mutant AraC-1Val genes are excised by partial EcoRI andXhoI digestion and cloned into the pCGN1761ENX plant expression plasmid.The expression cassettes containing 2×35S-Protox gene fusions areexcised by digestion with XbaI and cloned into the binary vectorpCIB200. These binary protox plasmids are transformed by electroporationinto Agrobacterium and then into Arabidopsis using the vacuuminfiltration method (Bechtold et al., 1993). Transformants are selectedon kanamycin, and T2 seed is generated from a number of independentlines. This seed is plated on GM media containing various concentrationsof protox-inhibiting herbicide and scored for germination and survival.Multiple transgenic lines overexpressing either the wild type or theresistant mutant protox produce significant numbers of green seedlingson an herbicide concentration that is lethal to the empty vectorcontrol.

Various modifications of the invention described herein will becomeapparent to those skilled in the art. Such modifications are intended tofall within the scope of the appended claims.

12 1719 base pairs nucleic acid single linear cDNA NO NO unknown CDS31..1644 /note= “Arabidopsis protox-1 cDNA; sequence from pWDC-2” 1TGACAAAATT CCGAATTCTC TGCGATTTCC ATG GAG TTA TCT CTT CTC CGT CCG 54 MetGlu Leu Ser Leu Leu Arg Pro 1 5 ACG ACT CAA TCG CTT CTT CCG TCG TTT TCGAAG CCC AAT CTC CGA TTA 102 Thr Thr Gln Ser Leu Leu Pro Ser Phe Ser LysPro Asn Leu Arg Leu 10 15 20 AAT GTT TAT AAG CCT CTT AGA CTC CGT TGT TCAGTG GCC GGT GGA CCA 150 Asn Val Tyr Lys Pro Leu Arg Leu Arg Cys Ser ValAla Gly Gly Pro 25 30 35 40 ACC GTC GGA TCT TCA AAA ATC GAA GGC GGA GGAGGC ACC ACC ATC ACG 198 Thr Val Gly Ser Ser Lys Ile Glu Gly Gly Gly GlyThr Thr Ile Thr 45 50 55 ACG GAT TGT GTG ATT GTC GGC GGA GGT ATT AGT GGTCTT TGC ATC GCT 246 Thr Asp Cys Val Ile Val Gly Gly Gly Ile Ser Gly LeuCys Ile Ala 60 65 70 CAG GCG CTT GCT ACT AAG CAT CCT GAT GCT GCT CCG AATTTA ATT GTG 294 Gln Ala Leu Ala Thr Lys His Pro Asp Ala Ala Pro Asn LeuIle Val 75 80 85 ACC GAG GCT AAG GAT CGT GTT GGA GGC AAC ATT ATC ACT CGTGAA GAG 342 Thr Glu Ala Lys Asp Arg Val Gly Gly Asn Ile Ile Thr Arg GluGlu 90 95 100 AAT GGT TTT CTC TGG GAA GAA GGT CCC AAT AGT TTT CAA CCGTCT GAT 390 Asn Gly Phe Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro SerAsp 105 110 115 120 CCT ATG CTC ACT ATG GTG GTA GAT AGT GGT TTG AAG GATGAT TTG GTG 438 Pro Met Leu Thr Met Val Val Asp Ser Gly Leu Lys Asp AspLeu Val 125 130 135 TTG GGA GAT CCT ACT GCG CCA AGG TTT GTG TTG TGG AATGGG AAA TTG 486 Leu Gly Asp Pro Thr Ala Pro Arg Phe Val Leu Trp Asn GlyLys Leu 140 145 150 AGG CCG GTT CCA TCG AAG CTA ACA GAC TTA CCG TTC TTTGAT TTG ATG 534 Arg Pro Val Pro Ser Lys Leu Thr Asp Leu Pro Phe Phe AspLeu Met 155 160 165 AGT ATT GGT GGG AAG ATT AGA GCT GGT TTT GGT GCA CTTGGC ATT CGA 582 Ser Ile Gly Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu GlyIle Arg 170 175 180 CCG TCA CCT CCA GGT CGT GAA GAA TCT GTG GAG GAG TTTGTA CGG CGT 630 Pro Ser Pro Pro Gly Arg Glu Glu Ser Val Glu Glu Phe ValArg Arg 185 190 195 200 AAC CTC GGT GAT GAG GTT TTT GAG CGC CTG ATT GAACCG TTT TGT TCA 678 Asn Leu Gly Asp Glu Val Phe Glu Arg Leu Ile Glu ProPhe Cys Ser 205 210 215 GGT GTT TAT GCT GGT GAT CCT TCA AAA CTG AGC ATGAAA GCA GCG TTT 726 Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met LysAla Ala Phe 220 225 230 GGG AAG GTT TGG AAA CTA GAG CAA AAT GGT GGA AGCATA ATA GGT GGT 774 Gly Lys Val Trp Lys Leu Glu Gln Asn Gly Gly Ser IleIle Gly Gly 235 240 245 ACT TTT AAG GCA ATT CAG GAG AGG AAA AAC GCT CCCAAG GCA GAA CGA 822 Thr Phe Lys Ala Ile Gln Glu Arg Lys Asn Ala Pro LysAla Glu Arg 250 255 260 GAC CCG CGC CTG CCA AAA CCA CAG GGC CAA ACA GTTGGT TCT TTC AGG 870 Asp Pro Arg Leu Pro Lys Pro Gln Gly Gln Thr Val GlySer Phe Arg 265 270 275 280 AAG GGA CTT CGA ATG TTG CCA GAA GCA ATA TCTGCA AGA TTA GGT AGC 918 Lys Gly Leu Arg Met Leu Pro Glu Ala Ile Ser AlaArg Leu Gly Ser 285 290 295 AAA GTT AAG TTG TCT TGG AAG CTC TCA GGT ATCACT AAG CTG GAG AGC 966 Lys Val Lys Leu Ser Trp Lys Leu Ser Gly Ile ThrLys Leu Glu Ser 300 305 310 GGA GGA TAC AAC TTA ACA TAT GAG ACT CCA GATGGT TTA GTT TCC GTG 1014 Gly Gly Tyr Asn Leu Thr Tyr Glu Thr Pro Asp GlyLeu Val Ser Val 315 320 325 CAG AGC AAA AGT GTT GTA ATG ACG GTG CCA TCTCAT GTT GCA AGT GGT 1062 Gln Ser Lys Ser Val Val Met Thr Val Pro Ser HisVal Ala Ser Gly 330 335 340 CTC TTG CGC CCT CTT TCT GAA TCT GCT GCA AATGCA CTC TCA AAA CTA 1110 Leu Leu Arg Pro Leu Ser Glu Ser Ala Ala Asn AlaLeu Ser Lys Leu 345 350 355 360 TAT TAC CCA CCA GTT GCA GCA GTA TCT ATCTCG TAC CCG AAA GAA GCA 1158 Tyr Tyr Pro Pro Val Ala Ala Val Ser Ile SerTyr Pro Lys Glu Ala 365 370 375 ATC CGA ACA GAA TGT TTG ATA GAT GGT GAACTA AAG GGT TTT GGG CAA 1206 Ile Arg Thr Glu Cys Leu Ile Asp Gly Glu LeuLys Gly Phe Gly Gln 380 385 390 TTG CAT CCA CGC ACG CAA GGA GTT GAA ACATTA GGA ACT ATC TAC AGC 1254 Leu His Pro Arg Thr Gln Gly Val Glu Thr LeuGly Thr Ile Tyr Ser 395 400 405 TCC TCA CTC TTT CCA AAT CGC GCA CCG CCCGGA AGA ATT TTG CTG TTG 1302 Ser Ser Leu Phe Pro Asn Arg Ala Pro Pro GlyArg Ile Leu Leu Leu 410 415 420 AAC TAC ATT GGC GGG TCT ACA AAC ACC GGAATT CTG TCC AAG TCT GAA 1350 Asn Tyr Ile Gly Gly Ser Thr Asn Thr Gly IleLeu Ser Lys Ser Glu 425 430 435 440 GGT GAG TTA GTG GAA GCA GTT GAC AGAGAT TTG AGG AAA ATG CTA ATT 1398 Gly Glu Leu Val Glu Ala Val Asp Arg AspLeu Arg Lys Met Leu Ile 445 450 455 AAG CCT AAT TCG ACC GAT CCA CTT AAATTA GGA GTT AGG GTA TGG CCT 1446 Lys Pro Asn Ser Thr Asp Pro Leu Lys LeuGly Val Arg Val Trp Pro 460 465 470 CAA GCC ATT CCT CAG TTT CTA GTT GGTCAC TTT GAT ATC CTT GAC ACG 1494 Gln Ala Ile Pro Gln Phe Leu Val Gly HisPhe Asp Ile Leu Asp Thr 475 480 485 GCT AAA TCA TCT CTA ACG TCT TCG GGCTAC GAA GGG CTA TTT TTG GGT 1542 Ala Lys Ser Ser Leu Thr Ser Ser Gly TyrGlu Gly Leu Phe Leu Gly 490 495 500 GGC AAT TAC GTC GCT GGT GTA GCC TTAGGC CGG TGT GTA GAA GGC GCA 1590 Gly Asn Tyr Val Ala Gly Val Ala Leu GlyArg Cys Val Glu Gly Ala 505 510 515 520 TAT GAA ACC GCG ATT GAG GTC AACAAC TTC ATG TCA CGG TAC GCT TAC 1638 Tyr Glu Thr Ala Ile Glu Val Asn AsnPhe Met Ser Arg Tyr Ala Tyr 525 530 535 AAG TAAATGTAAA ACATTAAATCTCCCAGCTTG CGTGAGTTTT ATTAAATATT 1691 Lys TTGAGATATC CAAAAAAAAA AAAAAAAA1719 537 amino acids amino acid linear protein unknown 2 Met Glu Leu SerLeu Leu Arg Pro Thr Thr Gln Ser Leu Leu Pro Ser 1 5 10 15 Phe Ser LysPro Asn Leu Arg Leu Asn Val Tyr Lys Pro Leu Arg Leu 20 25 30 Arg Cys SerVal Ala Gly Gly Pro Thr Val Gly Ser Ser Lys Ile Glu 35 40 45 Gly Gly GlyGly Thr Thr Ile Thr Thr Asp Cys Val Ile Val Gly Gly 50 55 60 Gly Ile SerGly Leu Cys Ile Ala Gln Ala Leu Ala Thr Lys His Pro 65 70 75 80 Asp AlaAla Pro Asn Leu Ile Val Thr Glu Ala Lys Asp Arg Val Gly 85 90 95 Gly AsnIle Ile Thr Arg Glu Glu Asn Gly Phe Leu Trp Glu Glu Gly 100 105 110 ProAsn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr Met Val Val Asp 115 120 125Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala Pro Arg 130 135140 Phe Val Leu Trp Asn Gly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr 145150 155 160 Asp Leu Pro Phe Phe Asp Leu Met Ser Ile Gly Gly Lys Ile ArgAla 165 170 175 Gly Phe Gly Ala Leu Gly Ile Arg Pro Ser Pro Pro Gly ArgGlu Glu 180 185 190 Ser Val Glu Glu Phe Val Arg Arg Asn Leu Gly Asp GluVal Phe Glu 195 200 205 Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr AlaGly Asp Pro Ser 210 215 220 Lys Leu Ser Met Lys Ala Ala Phe Gly Lys ValTrp Lys Leu Glu Gln 225 230 235 240 Asn Gly Gly Ser Ile Ile Gly Gly ThrPhe Lys Ala Ile Gln Glu Arg 245 250 255 Lys Asn Ala Pro Lys Ala Glu ArgAsp Pro Arg Leu Pro Lys Pro Gln 260 265 270 Gly Gln Thr Val Gly Ser PheArg Lys Gly Leu Arg Met Leu Pro Glu 275 280 285 Ala Ile Ser Ala Arg LeuGly Ser Lys Val Lys Leu Ser Trp Lys Leu 290 295 300 Ser Gly Ile Thr LysLeu Glu Ser Gly Gly Tyr Asn Leu Thr Tyr Glu 305 310 315 320 Thr Pro AspGly Leu Val Ser Val Gln Ser Lys Ser Val Val Met Thr 325 330 335 Val ProSer His Val Ala Ser Gly Leu Leu Arg Pro Leu Ser Glu Ser 340 345 350 AlaAla Asn Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val 355 360 365Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg Thr Glu Cys Leu Ile Asp 370 375380 Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Gly Val 385390 395 400 Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn ArgAla 405 410 415 Pro Pro Gly Arg Ile Leu Leu Leu Asn Tyr Ile Gly Gly SerThr Asn 420 425 430 Thr Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val GluAla Val Asp 435 440 445 Arg Asp Leu Arg Lys Met Leu Ile Lys Pro Asn SerThr Asp Pro Leu 450 455 460 Lys Leu Gly Val Arg Val Trp Pro Gln Ala IlePro Gln Phe Leu Val 465 470 475 480 Gly His Phe Asp Ile Leu Asp Thr AlaLys Ser Ser Leu Thr Ser Ser 485 490 495 Gly Tyr Glu Gly Leu Phe Leu GlyGly Asn Tyr Val Ala Gly Val Ala 500 505 510 Leu Gly Arg Cys Val Glu GlyAla Tyr Glu Thr Ala Ile Glu Val Asn 515 520 525 Asn Phe Met Ser Arg TyrAla Tyr Lys 530 535 1738 base pairs nucleic acid single linear cDNA NONO unknown CDS 70..1596 /note= “Arabidopsis protox-2 cDNA; sequence frompWDC-1” 3 TTTTTTACTT ATTTCCGTCA CTGCTTTCGA CTGGTCAGAG ATTTTGACTCTGAATTGTTG 60 CAGATAGCA ATG GCG TCT GGA GCA GTA GCA GAT CAT CAA ATT GAAGCG 108 Met Ala Ser Gly Ala Val Ala Asp His Gln Ile Glu Ala 1 5 10 GTTTCA GGA AAA AGA GTC GCA GTC GTA GGT GCA GGT GTA AGT GGA CTT 156 Val SerGly Lys Arg Val Ala Val Val Gly Ala Gly Val Ser Gly Leu 15 20 25 GCG GCGGCT TAC AAG TTG AAA TCG AGG GGT TTG AAT GTG ACT GTG TTT 204 Ala Ala AlaTyr Lys Leu Lys Ser Arg Gly Leu Asn Val Thr Val Phe 30 35 40 45 GAA GCTGAT GGA AGA GTA GGT GGG AAG TTG AGA AGT GTT ATG CAA AAT 252 Glu Ala AspGly Arg Val Gly Gly Lys Leu Arg Ser Val Met Gln Asn 50 55 60 GGT TTG ATTTGG GAT GAA GGA GCA AAC ACC ATG ACT GAG GCT GAG CCA 300 Gly Leu Ile TrpAsp Glu Gly Ala Asn Thr Met Thr Glu Ala Glu Pro 65 70 75 GAA GTT GGG AGTTTA CTT GAT GAT CTT GGG CTT CGT GAG AAA CAA CAA 348 Glu Val Gly Ser LeuLeu Asp Asp Leu Gly Leu Arg Glu Lys Gln Gln 80 85 90 TTT CCA ATT TCA CAGAAA AAG CGG TAT ATT GTG CGG AAT GGT GTA CCT 396 Phe Pro Ile Ser Gln LysLys Arg Tyr Ile Val Arg Asn Gly Val Pro 95 100 105 GTG ATG CTA CCT ACCAAT CCC ATA GAG CTG GTC ACA AGT AGT GTG CTC 444 Val Met Leu Pro Thr AsnPro Ile Glu Leu Val Thr Ser Ser Val Leu 110 115 120 125 TCT ACC CAA TCTAAG TTT CAA ATC TTG TTG GAA CCA TTT TTA TGG AAG 492 Ser Thr Gln Ser LysPhe Gln Ile Leu Leu Glu Pro Phe Leu Trp Lys 130 135 140 AAA AAG TCC TCAAAA GTC TCA GAT GCA TCT GCT GAA GAA AGT GTA AGC 540 Lys Lys Ser Ser LysVal Ser Asp Ala Ser Ala Glu Glu Ser Val Ser 145 150 155 GAG TTC TTT CAACGC CAT TTT GGA CAA GAG GTT GTT GAC TAT CTC ATC 588 Glu Phe Phe Gln ArgHis Phe Gly Gln Glu Val Val Asp Tyr Leu Ile 160 165 170 GAC CCT TTT GTTGGT GGA ACA AGT GCT GCG GAC CCT GAT TCC CTT TCA 636 Asp Pro Phe Val GlyGly Thr Ser Ala Ala Asp Pro Asp Ser Leu Ser 175 180 185 ATG AAG CAT TCTTTC CCA GAT CTC TGG AAT GTA GAG AAA AGT TTT GGC 684 Met Lys His Ser PhePro Asp Leu Trp Asn Val Glu Lys Ser Phe Gly 190 195 200 205 TCT ATT ATAGTC GGT GCA ATC AGA ACA AAG TTT GCT GCT AAA GGT GGT 732 Ser Ile Ile ValGly Ala Ile Arg Thr Lys Phe Ala Ala Lys Gly Gly 210 215 220 AAA AGT AGAGAC ACA AAG AGT TCT CCT GGC ACA AAA AAG GGT TCG CGT 780 Lys Ser Arg AspThr Lys Ser Ser Pro Gly Thr Lys Lys Gly Ser Arg 225 230 235 GGG TCA TTCTCT TTT AAG GGG GGA ATG CAG ATT CTT CCT GAT ACG TTG 828 Gly Ser Phe SerPhe Lys Gly Gly Met Gln Ile Leu Pro Asp Thr Leu 240 245 250 TGC AAA AGTCTC TCA CAT GAT GAG ATC AAT TTA GAC TCC AAG GTA CTC 876 Cys Lys Ser LeuSer His Asp Glu Ile Asn Leu Asp Ser Lys Val Leu 255 260 265 TCT TTG TCTTAC AAT TCT GGA TCA AGA CAG GAG AAC TGG TCA TTA TCT 924 Ser Leu Ser TyrAsn Ser Gly Ser Arg Gln Glu Asn Trp Ser Leu Ser 270 275 280 285 TGT GTTTCG CAT AAT GAA ACG CAG AGA CAA AAC CCC CAT TAT GAT GCT 972 Cys Val SerHis Asn Glu Thr Gln Arg Gln Asn Pro His Tyr Asp Ala 290 295 300 GTA ATTATG ACG GCT CCT CTG TGC AAT GTG AAG GAG ATG AAG GTT ATG 1020 Val Ile MetThr Ala Pro Leu Cys Asn Val Lys Glu Met Lys Val Met 305 310 315 AAA GGAGGA CAA CCC TTT CAG CTA AAC TTT CTC CCC GAG ATT AAT TAC 1068 Lys Gly GlyGln Pro Phe Gln Leu Asn Phe Leu Pro Glu Ile Asn Tyr 320 325 330 ATG CCCCTC TCG GTT TTA ATC ACC ACA TTC ACA AAG GAG AAA GTA AAG 1116 Met Pro LeuSer Val Leu Ile Thr Thr Phe Thr Lys Glu Lys Val Lys 335 340 345 AGA CCTCTT GAA GGC TTT GGG GTA CTC ATT CCA TCT AAG GAG CAA AAG 1164 Arg Pro LeuGlu Gly Phe Gly Val Leu Ile Pro Ser Lys Glu Gln Lys 350 355 360 365 CATGGT TTC AAA ACT CTA GGT ACA CTT TTT TCA TCA ATG ATG TTT CCA 1212 His GlyPhe Lys Thr Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro 370 375 380 GATCGT TCC CCT AGT GAC GTT CAT CTA TAT ACA ACT TTT ATT GGT GGG 1260 Asp ArgSer Pro Ser Asp Val His Leu Tyr Thr Thr Phe Ile Gly Gly 385 390 395 AGTAGG AAC CAG GAA CTA GCC AAA GCT TCC ACT GAC GAA TTA AAA CAA 1308 Ser ArgAsn Gln Glu Leu Ala Lys Ala Ser Thr Asp Glu Leu Lys Gln 400 405 410 GTTGTG ACT TCT GAC CTT CAG CGA CTG TTG GGG GTT GAA GGT GAA CCC 1356 Val ValThr Ser Asp Leu Gln Arg Leu Leu Gly Val Glu Gly Glu Pro 415 420 425 GTGTCT GTC AAC CAT TAC TAT TGG AGG AAA GCA TTC CCG TTG TAT GAC 1404 Val SerVal Asn His Tyr Tyr Trp Arg Lys Ala Phe Pro Leu Tyr Asp 430 435 440 445AGC AGC TAT GAC TCA GTC ATG GAA GCA ATT GAC AAG ATG GAG AAT GAT 1452 SerSer Tyr Asp Ser Val Met Glu Ala Ile Asp Lys Met Glu Asn Asp 450 455 460CTA CCT GGG TTC TTC TAT GCA GGT AAT CAT CGA GGG GGG CTC TCT GTT 1500 LeuPro Gly Phe Phe Tyr Ala Gly Asn His Arg Gly Gly Leu Ser Val 465 470 475GGG AAA TCA ATA GCA TCA GGT TGC AAA GCA GCT GAC CTT GTG ATC TCA 1548 GlyLys Ser Ile Ala Ser Gly Cys Lys Ala Ala Asp Leu Val Ile Ser 480 485 490TAC CTG GAG TCT TGC TCA AAT GAC AAG AAA CCA AAT GAC AGC TTA TAACATTG1603Tyr Leu Glu Ser Cys Ser Asn Asp Lys Lys Pro Asn Asp Ser Leu 495 500 505AAGGTTCGTC CCTTTTTATC ACTTACTTTG TAAACTTGTA AAATGCAACA AGCCGCCGTG 1663CGATTAGCCA ACAACTCAGC AAAACCCAGA TTCTCATAAG GCTCACTAAT TCCAGAATAA 1723ACTATTTATG TAAAA 1738 508 amino acids amino acid linear protein unknown4 Met Ala Ser Gly Ala Val Ala Asp His Gln Ile Glu Ala Val Ser Gly 1 5 1015 Lys Arg Val Ala Val Val Gly Ala Gly Val Ser Gly Leu Ala Ala Ala 20 2530 Tyr Lys Leu Lys Ser Arg Gly Leu Asn Val Thr Val Phe Glu Ala Asp 35 4045 Gly Arg Val Gly Gly Lys Leu Arg Ser Val Met Gln Asn Gly Leu Ile 50 5560 Trp Asp Glu Gly Ala Asn Thr Met Thr Glu Ala Glu Pro Glu Val Gly 65 7075 80 Ser Leu Leu Asp Asp Leu Gly Leu Arg Glu Lys Gln Gln Phe Pro Ile 8590 95 Ser Gln Lys Lys Arg Tyr Ile Val Arg Asn Gly Val Pro Val Met Leu100 105 110 Pro Thr Asn Pro Ile Glu Leu Val Thr Ser Ser Val Leu Ser ThrGln 115 120 125 Ser Lys Phe Gln Ile Leu Leu Glu Pro Phe Leu Trp Lys LysLys Ser 130 135 140 Ser Lys Val Ser Asp Ala Ser Ala Glu Glu Ser Val SerGlu Phe Phe 145 150 155 160 Gln Arg His Phe Gly Gln Glu Val Val Asp TyrLeu Ile Asp Pro Phe 165 170 175 Val Gly Gly Thr Ser Ala Ala Asp Pro AspSer Leu Ser Met Lys His 180 185 190 Ser Phe Pro Asp Leu Trp Asn Val GluLys Ser Phe Gly Ser Ile Ile 195 200 205 Val Gly Ala Ile Arg Thr Lys PheAla Ala Lys Gly Gly Lys Ser Arg 210 215 220 Asp Thr Lys Ser Ser Pro GlyThr Lys Lys Gly Ser Arg Gly Ser Phe 225 230 235 240 Ser Phe Lys Gly GlyMet Gln Ile Leu Pro Asp Thr Leu Cys Lys Ser 245 250 255 Leu Ser His AspGlu Ile Asn Leu Asp Ser Lys Val Leu Ser Leu Ser 260 265 270 Tyr Asn SerGly Ser Arg Gln Glu Asn Trp Ser Leu Ser Cys Val Ser 275 280 285 His AsnGlu Thr Gln Arg Gln Asn Pro His Tyr Asp Ala Val Ile Met 290 295 300 ThrAla Pro Leu Cys Asn Val Lys Glu Met Lys Val Met Lys Gly Gly 305 310 315320 Gln Pro Phe Gln Leu Asn Phe Leu Pro Glu Ile Asn Tyr Met Pro Leu 325330 335 Ser Val Leu Ile Thr Thr Phe Thr Lys Glu Lys Val Lys Arg Pro Leu340 345 350 Glu Gly Phe Gly Val Leu Ile Pro Ser Lys Glu Gln Lys His GlyPhe 355 360 365 Lys Thr Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro AspArg Ser 370 375 380 Pro Ser Asp Val His Leu Tyr Thr Thr Phe Ile Gly GlySer Arg Asn 385 390 395 400 Gln Glu Leu Ala Lys Ala Ser Thr Asp Glu LeuLys Gln Val Val Thr 405 410 415 Ser Asp Leu Gln Arg Leu Leu Gly Val GluGly Glu Pro Val Ser Val 420 425 430 Asn His Tyr Tyr Trp Arg Lys Ala PhePro Leu Tyr Asp Ser Ser Tyr 435 440 445 Asp Ser Val Met Glu Ala Ile AspLys Met Glu Asn Asp Leu Pro Gly 450 455 460 Phe Phe Tyr Ala Gly Asn HisArg Gly Gly Leu Ser Val Gly Lys Ser 465 470 475 480 Ile Ala Ser Gly CysLys Ala Ala Asp Leu Val Ile Ser Tyr Leu Glu 485 490 495 Ser Cys Ser AsnAsp Lys Lys Pro Asn Asp Ser Leu 500 505 1698 base pairs nucleic acidsingle linear cDNA NO NO unknown CDS 2..1453 /note= “Maize protox-1 cDNA(not full-length); sequence from pWDC-4” 5 G AAT TCG GCG GAC TGC GTC GTGGTG GGC GGA GGC ATC AGT GGC CTC 46 Asn Ser Ala Asp Cys Val Val Val GlyGly Gly Ile Ser Gly Leu 1 5 10 15 TGC ACC GCG CAG GCG CTG GCC ACG CGGCAC GGC GTC GGG GAC GTG CTT 94 Cys Thr Ala Gln Ala Leu Ala Thr Arg HisGly Val Gly Asp Val Leu 20 25 30 GTC ACG GAG GCC CGC GCC CGC CCC GGC GGCAAC ATT ACC ACC GTC GAG 142 Val Thr Glu Ala Arg Ala Arg Pro Gly Gly AsnIle Thr Thr Val Glu 35 40 45 CGC CCC GAG GAA GGG TAC CTC TGG GAG GAG GGTCCC AAC AGC TTC CAG 190 Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly ProAsn Ser Phe Gln 50 55 60 CCC TCC GAC CCC GTT CTC ACC ATG GCC GTG GAC AGCGGA CTG AAG GAT 238 Pro Ser Asp Pro Val Leu Thr Met Ala Val Asp Ser GlyLeu Lys Asp 65 70 75 GAC TTG GTT TTT GGG GAC CCA AAC GCG CCG CGT TTC GTGCTG TGG GAG 286 Asp Leu Val Phe Gly Asp Pro Asn Ala Pro Arg Phe Val LeuTrp Glu 80 85 90 95 GGG AAG CTG AGG CCC GTG CCA TCC AAG CCC GCC GAC CTCCCG TTC TTC 334 Gly Lys Leu Arg Pro Val Pro Ser Lys Pro Ala Asp Leu ProPhe Phe 100 105 110 GAT CTC ATG AGC ATC CCA GGG AAG CTC AGG GCC GGT CTAGGC GCG CTT 382 Asp Leu Met Ser Ile Pro Gly Lys Leu Arg Ala Gly Leu GlyAla Leu 115 120 125 GGC ATC CGC CCG CCT CCT CCA GGC CGC GAA GAG TCA GTGGAG GAG TTC 430 Gly Ile Arg Pro Pro Pro Pro Gly Arg Glu Glu Ser Val GluGlu Phe 130 135 140 GTG CGC CGC AAC CTC GGT GCT GAG GTC TTT GAG CGC CTCATT GAG CCT 478 Val Arg Arg Asn Leu Gly Ala Glu Val Phe Glu Arg Leu IleGlu Pro 145 150 155 TTC TGC TCA GGT GTC TAT GCT GGT GAT CCT TCT AAG CTCAGC ATG AAG 526 Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu SerMet Lys 160 165 170 175 GCT GCA TTT GGG AAG GTT TGG CGG TTG GAA GAA ACTGGA GGT AGT ATT 574 Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Glu Thr GlyGly Ser Ile 180 185 190 ATT GGT GGA ACC ATC AAG ACA ATT CAG GAG AGG AGCAAG AAT CCA AAA 622 Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Ser LysAsn Pro Lys 195 200 205 CCA CCG AGG GAT GCC CGC CTT CCG AAG CCA AAA GGGCAG ACA GTT GCA 670 Pro Pro Arg Asp Ala Arg Leu Pro Lys Pro Lys Gly GlnThr Val Ala 210 215 220 TCT TTC AGG AAG GGT CTT GCC ATG CTT CCA AAT GCCATT ACA TCC AGC 718 Ser Phe Arg Lys Gly Leu Ala Met Leu Pro Asn Ala IleThr Ser Ser 225 230 235 TTG GGT AGT AAA GTC AAA CTA TCA TGG AAA CTC ACGAGC ATT ACA AAA 766 Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu Thr SerIle Thr Lys 240 245 250 255 TCA GAT GAC AAG GGA TAT GTT TTG GAG TAT GAAACG CCA GAA GGG GTT 814 Ser Asp Asp Lys Gly Tyr Val Leu Glu Tyr Glu ThrPro Glu Gly Val 260 265 270 GTT TCG GTG CAG GCT AAA AGT GTT ATC ATG ACTATT CCA TCA TAT GTT 862 Val Ser Val Gln Ala Lys Ser Val Ile Met Thr IlePro Ser Tyr Val 275 280 285 GCT AGC AAC ATT TTG CGT CCA CTT TCA AGC GATGCT GCA GAT GCT CTA 910 Ala Ser Asn Ile Leu Arg Pro Leu Ser Ser Asp AlaAla Asp Ala Leu 290 295 300 TCA AGA TTC TAT TAT CCA CCG GTT GCT GCT GTAACT GTT TCG TAT CCA 958 Ser Arg Phe Tyr Tyr Pro Pro Val Ala Ala Val ThrVal Ser Tyr Pro 305 310 315 AAG GAA GCA ATT AGA AAA GAA TGC TTA ATT GATGGG GAA CTC CAG GGC 1006 Lys Glu Ala Ile Arg Lys Glu Cys Leu Ile Asp GlyGlu Leu Gln Gly 320 325 330 335 TTT GGC CAG TTG CAT CCA CGT AGT CAA GGAGTT GAG ACA TTA GGA ACA 1054 Phe Gly Gln Leu His Pro Arg Ser Gln Gly ValGlu Thr Leu Gly Thr 340 345 350 ATA TAC AGT TCC TCA CTC TTT CCA AAT CGTGCT CCT GAC GGT AGG GTG 1102 Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg AlaPro Asp Gly Arg Val 355 360 365 TTA CTT CTA AAC TAC ATA GGA GGT GCT ACAAAC ACA GGA ATT GTT TCC 1150 Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr AsnThr Gly Ile Val Ser 370 375 380 AAG ACT GAA AGT GAG CTG GTC GAA GCA GTTGAC CGT GAC CTC CGA AAA 1198 Lys Thr Glu Ser Glu Leu Val Glu Ala Val AspArg Asp Leu Arg Lys 385 390 395 ATG CTT ATA AAT TCT ACA GCA GTG GAC CCTTTA GTC CTT GGT GTT CGA 1246 Met Leu Ile Asn Ser Thr Ala Val Asp Pro LeuVal Leu Gly Val Arg 400 405 410 415 GTT TGG CCA CAA GCC ATA CCT CAG TTCCTG GTA GGA CAT CTT GAT CTT 1294 Val Trp Pro Gln Ala Ile Pro Gln Phe LeuVal Gly His Leu Asp Leu 420 425 430 CTG GAA GCC GCA AAA GCT GCC CTG GACCGA GGT GGC TAC GAT GGG CTG 1342 Leu Glu Ala Ala Lys Ala Ala Leu Asp ArgGly Gly Tyr Asp Gly Leu 435 440 445 TTC CTA GGA GGG AAC TAT GTT GCA GGAGTT GCC CTG GGC AGA TGC GTT 1390 Phe Leu Gly Gly Asn Tyr Val Ala Gly ValAla Leu Gly Arg Cys Val 450 455 460 GAG GGC GCG TAT GAA AGT GCC TCG CAAATA TCT GAC TTC TTG ACC AAG 1438 Glu Gly Ala Tyr Glu Ser Ala Ser Gln IleSer Asp Phe Leu Thr Lys 465 470 475 TAT GCC TAC AAG TGATGAAAGAAGTGGAGCGC TACTTGTTAA TCGTTTATGT 1490 Tyr Ala Tyr Lys 480 TGCATAGATGAGGTGCCTCC GGGGAAAAAA AAGCTTGAAT AGTATTTTTT ATTCTTATTT 1550 TGTAAATTGCATTTCTGTTC TTTTTTCTAT CAGTAATTAG TTATATTTTA GTTCTGTAGG 1610 AGATTGTTCTGTTCACTGCC CTTCAAAAGA AATTTTATTT TTCATTCTTT TATGAGAGCT 1670 GTGCTACTTAAAAAAAAAAA AAAAAAAA 1698 483 amino acids amino acid linear proteinunknown 6 Asn Ser Ala Asp Cys Val Val Val Gly Gly Gly Ile Ser Gly LeuCys 1 5 10 15 Thr Ala Gln Ala Leu Ala Thr Arg His Gly Val Gly Asp ValLeu Val 20 25 30 Thr Glu Ala Arg Ala Arg Pro Gly Gly Asn Ile Thr Thr ValGlu Arg 35 40 45 Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro Asn Ser PheGln Pro 50 55 60 Ser Asp Pro Val Leu Thr Met Ala Val Asp Ser Gly Leu LysAsp Asp 65 70 75 80 Leu Val Phe Gly Asp Pro Asn Ala Pro Arg Phe Val LeuTrp Glu Gly 85 90 95 Lys Leu Arg Pro Val Pro Ser Lys Pro Ala Asp Leu ProPhe Phe Asp 100 105 110 Leu Met Ser Ile Pro Gly Lys Leu Arg Ala Gly LeuGly Ala Leu Gly 115 120 125 Ile Arg Pro Pro Pro Pro Gly Arg Glu Glu SerVal Glu Glu Phe Val 130 135 140 Arg Arg Asn Leu Gly Ala Glu Val Phe GluArg Leu Ile Glu Pro Phe 145 150 155 160 Cys Ser Gly Val Tyr Ala Gly AspPro Ser Lys Leu Ser Met Lys Ala 165 170 175 Ala Phe Gly Lys Val Trp ArgLeu Glu Glu Thr Gly Gly Ser Ile Ile 180 185 190 Gly Gly Thr Ile Lys ThrIle Gln Glu Arg Ser Lys Asn Pro Lys Pro 195 200 205 Pro Arg Asp Ala ArgLeu Pro Lys Pro Lys Gly Gln Thr Val Ala Ser 210 215 220 Phe Arg Lys GlyLeu Ala Met Leu Pro Asn Ala Ile Thr Ser Ser Leu 225 230 235 240 Gly SerLys Val Lys Leu Ser Trp Lys Leu Thr Ser Ile Thr Lys Ser 245 250 255 AspAsp Lys Gly Tyr Val Leu Glu Tyr Glu Thr Pro Glu Gly Val Val 260 265 270Ser Val Gln Ala Lys Ser Val Ile Met Thr Ile Pro Ser Tyr Val Ala 275 280285 Ser Asn Ile Leu Arg Pro Leu Ser Ser Asp Ala Ala Asp Ala Leu Ser 290295 300 Arg Phe Tyr Tyr Pro Pro Val Ala Ala Val Thr Val Ser Tyr Pro Lys305 310 315 320 Glu Ala Ile Arg Lys Glu Cys Leu Ile Asp Gly Glu Leu GlnGly Phe 325 330 335 Gly Gln Leu His Pro Arg Ser Gln Gly Val Glu Thr LeuGly Thr Ile 340 345 350 Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro AspGly Arg Val Leu 355 360 365 Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn ThrGly Ile Val Ser Lys 370 375 380 Thr Glu Ser Glu Leu Val Glu Ala Val AspArg Asp Leu Arg Lys Met 385 390 395 400 Leu Ile Asn Ser Thr Ala Val AspPro Leu Val Leu Gly Val Arg Val 405 410 415 Trp Pro Gln Ala Ile Pro GlnPhe Leu Val Gly His Leu Asp Leu Leu 420 425 430 Glu Ala Ala Lys Ala AlaLeu Asp Arg Gly Gly Tyr Asp Gly Leu Phe 435 440 445 Leu Gly Gly Asn TyrVal Ala Gly Val Ala Leu Gly Arg Cys Val Glu 450 455 460 Gly Ala Tyr GluSer Ala Ser Gln Ile Ser Asp Phe Leu Thr Lys Tyr 465 470 475 480 Ala TyrLys 2061 base pairs nucleic acid single linear cDNA NO NO unknown CDS64..1698 /note= “Maize protox-2 cDNA; sequence from pWDC-3” 7 CTCTCCTACCTCCACCTCCA CGACAACAAG CAAATCCCCA TCCAGTTCCA AACCCTAACT 60 CAA ATG CTCGCT TTG ACT GCC TCA GCC TCA TCC GCT TCG TCC CAT CCT 108 Met Leu Ala LeuThr Ala Ser Ala Ser Ser Ala Ser Ser His Pro 1 5 10 15 TAT CGC CAC GCCTCC GCG CAC ACT CGT CGC CCC CGC CTA CGT GCG GTC 156 Tyr Arg His Ala SerAla His Thr Arg Arg Pro Arg Leu Arg Ala Val 20 25 30 CTC GCG ATG GCG GGCTCC GAC GAC CCC CGT GCA GCG CCC GCC AGA TCG 204 Leu Ala Met Ala Gly SerAsp Asp Pro Arg Ala Ala Pro Ala Arg Ser 35 40 45 GTC GCC GTC GTC GGC GCCGGG GTC AGC GGG CTC GCG GCG GCG TAC AGG 252 Val Ala Val Val Gly Ala GlyVal Ser Gly Leu Ala Ala Ala Tyr Arg 50 55 60 CTC AGA CAG AGC GGC GTG AACGTA ACG GTG TTC GAA GCG GCC GAC AGG 300 Leu Arg Gln Ser Gly Val Asn ValThr Val Phe Glu Ala Ala Asp Arg 65 70 75 GCG GGA GGA AAG ATA CGG ACC AATTCC GAG GGC GGG TTT GTC TGG GAT 348 Ala Gly Gly Lys Ile Arg Thr Asn SerGlu Gly Gly Phe Val Trp Asp 80 85 90 95 GAA GGA GCT AAC ACC ATG ACA GAAGGT GAA TGG GAG GCC AGT AGA CTG 396 Glu Gly Ala Asn Thr Met Thr Glu GlyGlu Trp Glu Ala Ser Arg Leu 100 105 110 ATT GAT GAT CTT GGT CTA CAA GACAAA CAG CAG TAT CCT AAC TCC CAA 444 Ile Asp Asp Leu Gly Leu Gln Asp LysGln Gln Tyr Pro Asn Ser Gln 115 120 125 CAC AAG CGT TAC ATT GTC AAA GATGGA GCA CCA GCA CTG ATT CCT TCG 492 His Lys Arg Tyr Ile Val Lys Asp GlyAla Pro Ala Leu Ile Pro Ser 130 135 140 GAT CCC ATT TCG CTA ATG AAA AGCAGT GTT CTT TCG ACA AAA TCA AAG 540 Asp Pro Ile Ser Leu Met Lys Ser SerVal Leu Ser Thr Lys Ser Lys 145 150 155 ATT GCG TTA TTT TTT GAA CCA TTTCTC TAC AAG AAA GCT AAC ACA AGA 588 Ile Ala Leu Phe Phe Glu Pro Phe LeuTyr Lys Lys Ala Asn Thr Arg 160 165 170 175 AAC TCT GGA AAA GTG TCT GAGGAG CAC TTG AGT GAG AGT GTT GGG AGC 636 Asn Ser Gly Lys Val Ser Glu GluHis Leu Ser Glu Ser Val Gly Ser 180 185 190 TTC TGT GAA CGC CAC TTT GGAAGA GAA GTT GTT GAC TAT TTT GTT GAT 684 Phe Cys Glu Arg His Phe Gly ArgGlu Val Val Asp Tyr Phe Val Asp 195 200 205 CCA TTT GTA GCT GGA ACA AGTGCA GGA GAT CCA GAG TCA CTA TCT ATT 732 Pro Phe Val Ala Gly Thr Ser AlaGly Asp Pro Glu Ser Leu Ser Ile 210 215 220 CGT CAT GCA TTC CCA GCA TTGTGG AAT TTG GAA AGA AAG TAT GGT TCA 780 Arg His Ala Phe Pro Ala Leu TrpAsn Leu Glu Arg Lys Tyr Gly Ser 225 230 235 GTT ATT GTT GGT GCC ATC TTGTCT AAG CTA GCA GCT AAA GGT GAT CCA 828 Val Ile Val Gly Ala Ile Leu SerLys Leu Ala Ala Lys Gly Asp Pro 240 245 250 255 GTA AAG ACA AGA CAT GATTCA TCA GGG AAA AGA AGG AAT AGA CGA GTG 876 Val Lys Thr Arg His Asp SerSer Gly Lys Arg Arg Asn Arg Arg Val 260 265 270 TCG TTT TCA TTT CAT GGTGGA ATG CAG TCA CTA ATA AAT GCA CTT CAC 924 Ser Phe Ser Phe His Gly GlyMet Gln Ser Leu Ile Asn Ala Leu His 275 280 285 AAT GAA GTT GGA GAT GATAAT GTG AAG CTT GGT ACA GAA GTG TTG TCA 972 Asn Glu Val Gly Asp Asp AsnVal Lys Leu Gly Thr Glu Val Leu Ser 290 295 300 TTG GCA TGT ACA TTT GATGGA GTT CCT GCA CTA GGC AGG TGG TCA ATT 1020 Leu Ala Cys Thr Phe Asp GlyVal Pro Ala Leu Gly Arg Trp Ser Ile 305 310 315 TCT GTT GAT TCG AAG GATAGC GGT GAC AAG GAC CTT GCT AGT AAC CAA 1068 Ser Val Asp Ser Lys Asp SerGly Asp Lys Asp Leu Ala Ser Asn Gln 320 325 330 335 ACC TTT GAT GCT GTTATA ATG ACA GCT CCA TTG TCA AAT GTC CGG AGG 1116 Thr Phe Asp Ala Val IleMet Thr Ala Pro Leu Ser Asn Val Arg Arg 340 345 350 ATG AAG TTC ACC AAAGGT GGA GCT CCG GTT GTT CTT GAC TTT CTT CCT 1164 Met Lys Phe Thr Lys GlyGly Ala Pro Val Val Leu Asp Phe Leu Pro 355 360 365 AAG ATG GAT TAT CTACCA CTA TCT CTC ATG GTG ACT GCT TTT AAG AAG 1212 Lys Met Asp Tyr Leu ProLeu Ser Leu Met Val Thr Ala Phe Lys Lys 370 375 380 GAT GAT GTC AAG AAACCT CTG GAA GGA TTT GGG GTC TTA ATA CCT TAC 1260 Asp Asp Val Lys Lys ProLeu Glu Gly Phe Gly Val Leu Ile Pro Tyr 385 390 395 AAG GAA CAG CAA AAACAT GGT CTG AAA ACC CTT GGG ACT CTC TTT TCC 1308 Lys Glu Gln Gln Lys HisGly Leu Lys Thr Leu Gly Thr Leu Phe Ser 400 405 410 415 TCA ATG ATG TTCCCA GAT CGA GCT CCT GAT GAC CAA TAT TTA TAT ACA 1356 Ser Met Met Phe ProAsp Arg Ala Pro Asp Asp Gln Tyr Leu Tyr Thr 420 425 430 ACA TTT GTT GGGGGT AGC CAC AAT AGA GAT CTT GCT GGA GCT CCA ACG 1404 Thr Phe Val Gly GlySer His Asn Arg Asp Leu Ala Gly Ala Pro Thr 435 440 445 TCT ATT CTG AAACAA CTT GTG ACC TCT GAC CTT AAA AAA CTC TTG GGC 1452 Ser Ile Leu Lys GlnLeu Val Thr Ser Asp Leu Lys Lys Leu Leu Gly 450 455 460 GTA GAG GGG CAACCA ACT TTT GTC AAG CAT GTA TAC TGG GGA AAT GCT 1500 Val Glu Gly Gln ProThr Phe Val Lys His Val Tyr Trp Gly Asn Ala 465 470 475 TTT CCT TTG TATGGC CAT GAT TAT AGT TCT GTA TTG GAA GCT ATA GAA 1548 Phe Pro Leu Tyr GlyHis Asp Tyr Ser Ser Val Leu Glu Ala Ile Glu 480 485 490 495 AAG ATG GAGAAA AAC CTT CCA GGG TTC TTC TAC GCA GGA AAT AGC AAG 1596 Lys Met Glu LysAsn Leu Pro Gly Phe Phe Tyr Ala Gly Asn Ser Lys 500 505 510 GAT GGG CTTGCT GTT GGA AGT GTT ATA GCT TCA GGA AGC AAG GCT GCT 1644 Asp Gly Leu AlaVal Gly Ser Val Ile Ala Ser Gly Ser Lys Ala Ala 515 520 525 GAC CTT GCAATC TCA TAT CTT GAA TCT CAC ACC AAG CAT AAT AAT TCA 1692 Asp Leu Ala IleSer Tyr Leu Glu Ser His Thr Lys His Asn Asn Ser 530 535 540 CATTGAAAGTGTC TGACCTATCC TCTAGCAGTT GTCGACAAAT TTCTCCAGTT 1745 His 545CATGTACAGT AGAAACCGAT GCGTTGCAGT TTCAGAACAT CTTCACTTCT TCAGATATTA 1805ACCCTTCGTT GAACATCCAC CAGAAAGGTA GTCACATGTG TAAGTGGGAA AATGAGGTTA 1865AAAACTATTA TGGCGGCCGA AATGTTCCTT TTTGTTTTCC TCACAAGTGG CCTACGACAC 1925TTGATGTTGG AAATACATTT AAATTTGTTG AATTGTTTGA GAACACATGC GTGACGTGTA 1985ATATTTGCCT ATTGTGATTT TAGCAGTAGT CTTGGCCAGA TTATGCTTTA CGCCTTTAAA 2045AAAAAAAAAA AAAAAA 2061 544 amino acids amino acid linear protein unknown8 Met Leu Ala Leu Thr Ala Ser Ala Ser Ser Ala Ser Ser His Pro Tyr 1 5 1015 Arg His Ala Ser Ala His Thr Arg Arg Pro Arg Leu Arg Ala Val Leu 20 2530 Ala Met Ala Gly Ser Asp Asp Pro Arg Ala Ala Pro Ala Arg Ser Val 35 4045 Ala Val Val Gly Ala Gly Val Ser Gly Leu Ala Ala Ala Tyr Arg Leu 50 5560 Arg Gln Ser Gly Val Asn Val Thr Val Phe Glu Ala Ala Asp Arg Ala 65 7075 80 Gly Gly Lys Ile Arg Thr Asn Ser Glu Gly Gly Phe Val Trp Asp Glu 8590 95 Gly Ala Asn Thr Met Thr Glu Gly Glu Trp Glu Ala Ser Arg Leu Ile100 105 110 Asp Asp Leu Gly Leu Gln Asp Lys Gln Gln Tyr Pro Asn Ser GlnHis 115 120 125 Lys Arg Tyr Ile Val Lys Asp Gly Ala Pro Ala Leu Ile ProSer Asp 130 135 140 Pro Ile Ser Leu Met Lys Ser Ser Val Leu Ser Thr LysSer Lys Ile 145 150 155 160 Ala Leu Phe Phe Glu Pro Phe Leu Tyr Lys LysAla Asn Thr Arg Asn 165 170 175 Ser Gly Lys Val Ser Glu Glu His Leu SerGlu Ser Val Gly Ser Phe 180 185 190 Cys Glu Arg His Phe Gly Arg Glu ValVal Asp Tyr Phe Val Asp Pro 195 200 205 Phe Val Ala Gly Thr Ser Ala GlyAsp Pro Glu Ser Leu Ser Ile Arg 210 215 220 His Ala Phe Pro Ala Leu TrpAsn Leu Glu Arg Lys Tyr Gly Ser Val 225 230 235 240 Ile Val Gly Ala IleLeu Ser Lys Leu Ala Ala Lys Gly Asp Pro Val 245 250 255 Lys Thr Arg HisAsp Ser Ser Gly Lys Arg Arg Asn Arg Arg Val Ser 260 265 270 Phe Ser PheHis Gly Gly Met Gln Ser Leu Ile Asn Ala Leu His Asn 275 280 285 Glu ValGly Asp Asp Asn Val Lys Leu Gly Thr Glu Val Leu Ser Leu 290 295 300 AlaCys Thr Phe Asp Gly Val Pro Ala Leu Gly Arg Trp Ser Ile Ser 305 310 315320 Val Asp Ser Lys Asp Ser Gly Asp Lys Asp Leu Ala Ser Asn Gln Thr 325330 335 Phe Asp Ala Val Ile Met Thr Ala Pro Leu Ser Asn Val Arg Arg Met340 345 350 Lys Phe Thr Lys Gly Gly Ala Pro Val Val Leu Asp Phe Leu ProLys 355 360 365 Met Asp Tyr Leu Pro Leu Ser Leu Met Val Thr Ala Phe LysLys Asp 370 375 380 Asp Val Lys Lys Pro Leu Glu Gly Phe Gly Val Leu IlePro Tyr Lys 385 390 395 400 Glu Gln Gln Lys His Gly Leu Lys Thr Leu GlyThr Leu Phe Ser Ser 405 410 415 Met Met Phe Pro Asp Arg Ala Pro Asp AspGln Tyr Leu Tyr Thr Thr 420 425 430 Phe Val Gly Gly Ser His Asn Arg AspLeu Ala Gly Ala Pro Thr Ser 435 440 445 Ile Leu Lys Gln Leu Val Thr SerAsp Leu Lys Lys Leu Leu Gly Val 450 455 460 Glu Gly Gln Pro Thr Phe ValLys His Val Tyr Trp Gly Asn Ala Phe 465 470 475 480 Pro Leu Tyr Gly HisAsp Tyr Ser Ser Val Leu Glu Ala Ile Glu Lys 485 490 495 Met Glu Lys AsnLeu Pro Gly Phe Phe Tyr Ala Gly Asn Ser Lys Asp 500 505 510 Gly Leu AlaVal Gly Ser Val Ile Ala Ser Gly Ser Lys Ala Ala Asp 515 520 525 Leu AlaIle Ser Tyr Leu Glu Ser His Thr Lys His Asn Asn Ser His 530 535 540 1697base pairs nucleic acid single linear cDNA NO NO unknown CDS 29..1501/note= “Yeast protox-3 cDNA; sequence from pWDC-5” 9 TTGGCATTTGCCTTGAACCA ACAATTCT ATG TCA ATT GCA ATT TGT GGA GGA 52 Met Ser Ile AlaIle Cys Gly Gly 1 5 GGT ATA GCT GGT CTT AGT ACA GCA TTT TAT CTT GCT AGATTG ATT CCA 100 Gly Ile Ala Gly Leu Ser Thr Ala Phe Tyr Leu Ala Arg LeuIle Pro 10 15 20 AAA TGT ACT ATT GAT TTG TAC GAA AAA GGT CCT CGT TTA GGTGGA TGG 148 Lys Cys Thr Ile Asp Leu Tyr Glu Lys Gly Pro Arg Leu Gly GlyTrp 25 30 35 40 CTT CAG TCG GTC AAA ATC CCG TGT GCA GAT TCT CCA ACA GGAACG GTT 196 Leu Gln Ser Val Lys Ile Pro Cys Ala Asp Ser Pro Thr Gly ThrVal 45 50 55 TTG TTT GAG CAA GGT CCT AGA ACT CTT CGT CCT GCT GGG GTT GCTGGC 244 Leu Phe Glu Gln Gly Pro Arg Thr Leu Arg Pro Ala Gly Val Ala Gly60 65 70 TTA GCA AAC TTA GAT TTA ATT AGC AAG TTG GGC ATC GAA GAC AAG TTG292 Leu Ala Asn Leu Asp Leu Ile Ser Lys Leu Gly Ile Glu Asp Lys Leu 7580 85 TTA AGG ATT TCG AGC AAT TCT CCC AGC GCA AAA AAC CGA TAT ATT TAT340 Leu Arg Ile Ser Ser Asn Ser Pro Ser Ala Lys Asn Arg Tyr Ile Tyr 9095 100 TAC CCA GAT CGC TTA AAT GAA ATT CCT TCA AGC ATT TTA GGG AGT ATA388 Tyr Pro Asp Arg Leu Asn Glu Ile Pro Ser Ser Ile Leu Gly Ser Ile 105110 115 120 AAG TCG ATT ATG CAG CCT GCT TTG CGT CCG ATG CCT TTG GCT ATGATG 436 Lys Ser Ile Met Gln Pro Ala Leu Arg Pro Met Pro Leu Ala Met Met125 130 135 CTT GAG CCC TTT CGT AAA AGT AAG CGA GAT TCG ACA GAT GAA AGCGTG 484 Leu Glu Pro Phe Arg Lys Ser Lys Arg Asp Ser Thr Asp Glu Ser Val140 145 150 GGT TCA TTT ATG AGA AGA AGA TTT GGT AAA AAC GTT ACG GAT AGAGTT 532 Gly Ser Phe Met Arg Arg Arg Phe Gly Lys Asn Val Thr Asp Arg Val155 160 165 ATG AGT GCA ATG ATA AAT GGT ATT TAT GCT GGT GAT TTG AAT GATTTG 580 Met Ser Ala Met Ile Asn Gly Ile Tyr Ala Gly Asp Leu Asn Asp Leu170 175 180 TCT ATG CAT TCT AGC ATG TTT GGA TTT TTA GCG AAG ATT GAA AAAAAG 628 Ser Met His Ser Ser Met Phe Gly Phe Leu Ala Lys Ile Glu Lys Lys185 190 195 200 TAT GGA AAC ATT ACT TTG GGA TTA ATT AGA GCT CTT CTT GCACGT GAA 676 Tyr Gly Asn Ile Thr Leu Gly Leu Ile Arg Ala Leu Leu Ala ArgGlu 205 210 215 ATA TTA TCT CCT GCT GAG AAA GCT TTG GAA AGC AGC ACT ACTCGC AGA 724 Ile Leu Ser Pro Ala Glu Lys Ala Leu Glu Ser Ser Thr Thr ArgArg 220 225 230 GCC AAA AAC AGC AGA GCT GTC AAA CAG TAT GAA ATC GAC AAGTAT GTT 772 Ala Lys Asn Ser Arg Ala Val Lys Gln Tyr Glu Ile Asp Lys TyrVal 235 240 245 GCT TTC AAG GAA GGG ATT GAG ACT ATT ACA TTG TCA ATA GCAGAT GAA 820 Ala Phe Lys Glu Gly Ile Glu Thr Ile Thr Leu Ser Ile Ala AspGlu 250 255 260 TTA AAA AAA ATG CCG AAT GTC AAG ATA CAT CTA AAC AAA CCGGCC CAA 868 Leu Lys Lys Met Pro Asn Val Lys Ile His Leu Asn Lys Pro AlaGln 265 270 275 280 ACT TTG GTT CCA CAT AAA ACT CAG TCT CTT GTA GAC GTCAAT GGT CAA 916 Thr Leu Val Pro His Lys Thr Gln Ser Leu Val Asp Val AsnGly Gln 285 290 295 GCT TAC GAG TAT GTT GTG TTT GCA AAC TCT TCT CGC AATTTA GAG AAT 964 Ala Tyr Glu Tyr Val Val Phe Ala Asn Ser Ser Arg Asn LeuGlu Asn 300 305 310 CTA ATA TCT TGT CCT AAA ATG GAA ACT CCG ACG TCG AGTGTT TAT GTC 1012 Leu Ile Ser Cys Pro Lys Met Glu Thr Pro Thr Ser Ser ValTyr Val 315 320 325 GTC AAC GTT TAT TAT AAG GAC CCT AAT GTT CTT CCA ATCCGT GGT TTT 1060 Val Asn Val Tyr Tyr Lys Asp Pro Asn Val Leu Pro Ile ArgGly Phe 330 335 340 GGG CTT TTG ATT CCA TCA TGC ACT CCA AAT AAT CCG AATCAT GTT CTT 1108 Gly Leu Leu Ile Pro Ser Cys Thr Pro Asn Asn Pro Asn HisVal Leu 345 350 355 360 GGT ATC GTT TTT GAT AGT GAG CAA AAC AAC CCT GAAAAT GGA AGC AAG 1156 Gly Ile Val Phe Asp Ser Glu Gln Asn Asn Pro Glu AsnGly Ser Lys 365 370 375 GTC ACT GTC ATG ATG GGA GGG TCT GCT TAT ACA AAAAAT ACT TCT TTG 1204 Val Thr Val Met Met Gly Gly Ser Ala Tyr Thr Lys AsnThr Ser Leu 380 385 390 ATT CCA ACC AAC CCC GAA GAA GCC GTT AAC AAT GCTCTC AAA GCT TTG 1252 Ile Pro Thr Asn Pro Glu Glu Ala Val Asn Asn Ala LeuLys Ala Leu 395 400 405 CAG CAT ACT TTA AAA ATA TCC AGT AAG CCA ACA CTCACG AAT GCA ACA 1300 Gln His Thr Leu Lys Ile Ser Ser Lys Pro Thr Leu ThrAsn Ala Thr 410 415 420 TTA CAA CCA AAT TGC ATC CCT CAA TAT CGT GTT GGGCAT CAA GAT AAT 1348 Leu Gln Pro Asn Cys Ile Pro Gln Tyr Arg Val Gly HisGln Asp Asn 425 430 435 440 CTT AAT TCT TTG AAA TCT TGG ATT GAG AAA AATATG GGA GGG CGA ATT 1396 Leu Asn Ser Leu Lys Ser Trp Ile Glu Lys Asn MetGly Gly Arg Ile 445 450 455 CTT CTA ACT GGA AGT TGG TAT AAT GGT GTT AGTATT GGG GAT TGT ATT 1444 Leu Leu Thr Gly Ser Trp Tyr Asn Gly Val Ser IleGly Asp Cys Ile 460 465 470 ATG AAT GGA CAT TCA ACA GCT CGA AAA CTA GCATCA TTG ATG AAT TCT 1492 Met Asn Gly His Ser Thr Ala Arg Lys Leu Ala SerLeu Met Asn Ser 475 480 485 TCT TCT TGAGCGTTTA TAAATGTTGA TATAAAATTAGTATATAGTT CCTTTGATTA 1548 Ser Ser 490 TTTTATGAGT TGAAAATGCC ACTTGTGAAATAATTTTGCA CAAGCCCTTT TATTACAGAC 1608 GTATATGCGA GGACATTCGA CAAACGTTTGAAATTAAAAA TCATATGCCT TTTAGCTTAA 1668 GACATCAAGG TCATGAATAA TAAAATTTT1697 490 amino acids amino acid linear protein unknown 10 Met Ser IleAla Ile Cys Gly Gly Gly Ile Ala Gly Leu Ser Thr Ala 1 5 10 15 Phe TyrLeu Ala Arg Leu Ile Pro Lys Cys Thr Ile Asp Leu Tyr Glu 20 25 30 Lys GlyPro Arg Leu Gly Gly Trp Leu Gln Ser Val Lys Ile Pro Cys 35 40 45 Ala AspSer Pro Thr Gly Thr Val Leu Phe Glu Gln Gly Pro Arg Thr 50 55 60 Leu ArgPro Ala Gly Val Ala Gly Leu Ala Asn Leu Asp Leu Ile Ser 65 70 75 80 LysLeu Gly Ile Glu Asp Lys Leu Leu Arg Ile Ser Ser Asn Ser Pro 85 90 95 SerAla Lys Asn Arg Tyr Ile Tyr Tyr Pro Asp Arg Leu Asn Glu Ile 100 105 110Pro Ser Ser Ile Leu Gly Ser Ile Lys Ser Ile Met Gln Pro Ala Leu 115 120125 Arg Pro Met Pro Leu Ala Met Met Leu Glu Pro Phe Arg Lys Ser Lys 130135 140 Arg Asp Ser Thr Asp Glu Ser Val Gly Ser Phe Met Arg Arg Arg Phe145 150 155 160 Gly Lys Asn Val Thr Asp Arg Val Met Ser Ala Met Ile AsnGly Ile 165 170 175 Tyr Ala Gly Asp Leu Asn Asp Leu Ser Met His Ser SerMet Phe Gly 180 185 190 Phe Leu Ala Lys Ile Glu Lys Lys Tyr Gly Asn IleThr Leu Gly Leu 195 200 205 Ile Arg Ala Leu Leu Ala Arg Glu Ile Leu SerPro Ala Glu Lys Ala 210 215 220 Leu Glu Ser Ser Thr Thr Arg Arg Ala LysAsn Ser Arg Ala Val Lys 225 230 235 240 Gln Tyr Glu Ile Asp Lys Tyr ValAla Phe Lys Glu Gly Ile Glu Thr 245 250 255 Ile Thr Leu Ser Ile Ala AspGlu Leu Lys Lys Met Pro Asn Val Lys 260 265 270 Ile His Leu Asn Lys ProAla Gln Thr Leu Val Pro His Lys Thr Gln 275 280 285 Ser Leu Val Asp ValAsn Gly Gln Ala Tyr Glu Tyr Val Val Phe Ala 290 295 300 Asn Ser Ser ArgAsn Leu Glu Asn Leu Ile Ser Cys Pro Lys Met Glu 305 310 315 320 Thr ProThr Ser Ser Val Tyr Val Val Asn Val Tyr Tyr Lys Asp Pro 325 330 335 AsnVal Leu Pro Ile Arg Gly Phe Gly Leu Leu Ile Pro Ser Cys Thr 340 345 350Pro Asn Asn Pro Asn His Val Leu Gly Ile Val Phe Asp Ser Glu Gln 355 360365 Asn Asn Pro Glu Asn Gly Ser Lys Val Thr Val Met Met Gly Gly Ser 370375 380 Ala Tyr Thr Lys Asn Thr Ser Leu Ile Pro Thr Asn Pro Glu Glu Ala385 390 395 400 Val Asn Asn Ala Leu Lys Ala Leu Gln His Thr Leu Lys IleSer Ser 405 410 415 Lys Pro Thr Leu Thr Asn Ala Thr Leu Gln Pro Asn CysIle Pro Gln 420 425 430 Tyr Arg Val Gly His Gln Asp Asn Leu Asn Ser LeuLys Ser Trp Ile 435 440 445 Glu Lys Asn Met Gly Gly Arg Ile Leu Leu ThrGly Ser Trp Tyr Asn 450 455 460 Gly Val Ser Ile Gly Asp Cys Ile Met AsnGly His Ser Thr Ala Arg 465 470 475 480 Lys Leu Ala Ser Leu Met Asn SerSer Ser 485 490 41 base pairs nucleic acid single linear other nucleicacid oligonucleotide used to construct pCGN1761ENX NO NO unknown 11AATTATGACG TAACGTAGGA ATTAGCGGCC CGCTCTCGAG T 41 40 base pairs nucleicacid single linear other nucleic acid oligonucleotide used to constructpCGN1761ENX NO NO unknown 12 AATTACTCGA GAGCGGCCGC GAATTCCTAC GTTACGTCAT40

We claim:
 1. A probe which specifically hybridizes to a eukaryoticprotoporphyrinogen oxidase gene or mRNA, wherein said probe comprises acontiguous portion of the coding sequence for a protoporphyrinogenoxidase from a eukaryote at least 10 nucleotides in length.
 2. The probeof claim 1 wherein said coding sequence is selected from the groupconsisting of SEQ ID Nos. 1, 3, 5, 7 and
 9. 3. The probe of claim 1wherein said probe comprises a contiguous portion of the coding sequencefor a protoporphyrinogen oxidase gene or mRNA that is derived from aplant.
 4. The probe of claim 1 wherein said eukaryote is a plant.
 5. Amethod for identifying a eukaryotic protoporphyrinogen oxidase gene ormRNA comprising, obtaining a sample containing nucleic acid moleculesfrom a eukaryote, combining a probe of claim 1 with said sample underconditions wherein said probe binds specifically to a eukaryoticprotoporphyrinogen oxidase gene or mRNA, isolating said probe bound tosaid eukaryotic protoporphyrinogen oxidase gene or mRNA.
 6. The methodof claim 5 wherein said probe is derived from a coding sequence selectedfrom the group consisting of SEQ ID Nos. 1, 3, 5, 7 and
 9. 7. The methodof claim 5 wherein said probe comprises a contiguous portion of thecoding sequence for a protoporphyrinogen oxidase from a plant.